Monday, July 12, 2010

Klotho more than “ninphae”



        
    

 

From Greek mythology “Klotho” was one of three Moirae. She is responsible for spinning the thread of human life, so that she mades major decisons when a person is born. She controls who born through his life she also decide who has to be saved or put to death.

Two other sisters , Lachesis and Atropos, are responsible of human destiny and influence their misery and suffering.

Clotho assisted Hermes to create the alphabet, and forced the goddess Afrodite into making love with other gods, killed the Titan Typhon with poison fruits and persuaded Zeus to kill Asclepius with a bolt of lightning.

As you know Asclepius, the Roman Esculapius, is the god of Medicine and Healing. Asclepius has a daughter Epione ( Goddess of soothin of pain) and he his the father of : Hygieia (Hygiene), Panacea (Universal Remedy), Aceso (Goddess of Healing process), Leso or Laso (Goddess of recuperation from illness), Aglaea or Aglaia (Shining one, splendor, brillant, Healthy Glow) wife of Hephaesto and mother of Eucleia (good repute), Eupheme (Acclaim), Euthenia (Prosperity), Philophrosyne (Welcome).

The rod of Asclepius, a snake-entwined staff, remains a symbol of medicine today, also if sometimes a staff with two snakes (the caduceus) is mistakenly used instead.

He was one of the Apollo’s sons.

Asklepios

Asclepius

God of Medicine

Is KLOTHO, an anti-ageing hormone ?

The KLOTHO gene was identified serendipitously through a hypomorphic allele that results in severe early degenerative changes and short lifespan ( Nature 1997). The homozygous mutant animals develop normally until 3 weeks of age, then exhibit severe growth retardation, osteoporosis, ectopic calcification, aterosclerosis, emphysema and atrophy of the skin, thymus, testes and ovaries, and die at an average age of 61 days.

KLOTHO prolong lifespan at least in part by inhibiting insulin-IGF-1 signalling. KLOTHO may be secreted by KIDNEY cells blocking both IGF-1 and Insulin receptor action at adipocytes and target tissue levels.

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Levels of calcium and phosphate are elevated in KLOTHO deficient mice, demonstrating that this protein has a role in calcium and phoshate homeostasis. The altered calcium and phoshate levels are due to elevated 1,25 OH Vitamin D3 levels, which result from increased expression of 1alfa hydroxylase gene activity at kidney level.

Normalization of 1,25 OH Vitamin D3 with a Vitamin D deficient diet partially rescued some of the KLOTHO deficient phenotypes, including slow growth, ectopic calcification and early death. This suggests that the putative pro-ageing effect of KLTHO deficiency is not associated with ageing itself, but rather that the pathology is related to altered Vitamin D metabolism.

FGF23 KO mice share many features with the KLOTHO deficient mouse, including hypercalcemia, hyperphosphatemia, ectopic calcification, hypoglycaemia, infertility, and very short lifespan.

FGF23 is a circulating factor that is produced in the bone and inhibits phosphate transport in renal proximal tubular cells. FGF23 deficiency results in phosphate retention and hyperphosphatemia, constitutively elevated expression of 1 alfa hydroxylase, elevated levels of 1,25 OH Vitamin D3 and hypercalcaemia.

Interestingly without KLOTHO the functions of FGF23 is literally abolished.

Many tissues express FGF Receptors subtypes that interact with the KLOTHO-FGF23 complex, therefore it is possible that KLOTHO exerts his anti-ageing action through the activation of his enzymatic activity i.e. beta glucoronidation.

We know that all steroids enzymes including all lipophilic vitamins such as Vitamin D, with a steroid like structure, can be glucuronized in order to achieve a better hydrophility and flow into the blood vessels. The pool of glucuronide-linked steroids hormones is an inactive quote of hormones, those destiny is in normal condition to be metabolized further into hepatic cells or recycled by endocrine organs.

KLOTHO can hydrolyse STEROID GLUCURONIDES, including estradiol, estrone, estriol and vitamin D so that some effects of KLOTHO can occur through processing of inactive streroid glucuronides to active steroids hormones. Steroid hormones could have a role in the regulation of ageing in mammals.

 

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References

Arking DE, Krebsova A, Macek M Sr et al. Association of human aging with a functional variant of klotho. Proc Natl Acad Sci USA 2002;99:856-61.

Duce JA, Podvin S, Hollander W et al. Gene profiling analysis implicates klotho as an important contributor to aging changes in brain white matter of rhesus monkey. Glia 2008;56:106-117.

Arking DE, Becker DM, Yanek LR et al. KLOTHO allele status and the risk of early-onset occult coronary artery disease. Am J Hum Genet 2003;72:1154-61.

Mitani H, Ishizaka T, Aizawa T et al. In vivo klotho gene transfer ameliorates angiotensin II-induced renal damage. Hypertension 2002;39:838-43.

Ogata N, Matsumura Y, Shiraki M et al. Association of klotho gene polymorphism with bone density and spondylosis of the lumbar spine in postmenopausal women. Bone 2002;31:37-42.

Yamada Y, Ando F, Niino N et al. Association of polymorphisms of the androgen receptor and klotho genes with bone mineral density in Japanese women. J Mol Med 2005;83:50-7.

Mullin BH, Wilson SG, Islam FM etr al. Klotho gene polymorphisms are associated with osteocalcin levels but not bone density of aged postmenopausal women. Calcif Tissue Int 2005;77:145-51.

Kawano K, Ogata N, Chiano M et al. Klotho gene polymorphisms associated with bone density of aged postmenopausal women. J Bone Miner Res 2002;17:1744-51.

Kuro-o M, Matsumura Y, Aizawa H et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 1997;390:45-51.

Roth GS, Lane MA, Ingram DK et al. Biomarkers of caloric restriction may predict longevity in humans. Science 2002;297:811.

Kurosu H, Yamamoto M, Clark JD et al. Suppression of aging in mice by the hormone Klotho. Science 2005;309:1829-33.

Chang Q, Hoefs S, van der Kemp AW et al. The beta glucuronidase Klotho hydrolyzes and activates the TRPV5 channel. Science 2005;310:490-3.

Russell SJ, Kahn CR. Endocrine regulation of aging. Nat Rev Mol Cell Biol 2007;8:681-91.

Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and aging. Science 1996;273:59-63.

Ingram DK, Cutler RG, Weindruch R et al. Dietary restriction and aging: the initiation of a primate study. J Gerontol 1990;45:B148-B163.

Sohal RS, Argarwal S, Candas M et al. Effect of age and caloric restriction on DNA oxidative damage in different tissues of C57BL/6 mice. Mech Ageing Dev 1994;76:215-24.

Lee C-K, Klopp RG, Weindruch R et al. Gene expression profile of aging and its retardation by caloric restriction. Science 1999;285:1390-3.

Kujoth GC, Hiona A, Pugh TD et al. Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging. Science 2003;309:481-4.

Weindruch R, Sohal RS. Caloric intake and aging. N Engl J Med 1997;337:986-94.

Priè D, Friedlander G. Genetic disorders of renal phosphate transport. N Engl J Med 2010;362:2399-2409.

Monday, May 31, 2010

High Resolution peripheral Quantitative Tomography and bone quality.

 
  
 
 

Bisphosphonates

Bisphosphonates (BP) are synthetic analogues of inorganic pyrphosphate with a central carbon instead of an oxigen element that protect BPs from biological degradation.

P – O – P inorganic pyrophosphate

P – C – P bisphosphonates

Thos P-C-P backbone is identical across all types of BPs, and two sidechains account for their biological diversity: a hydroxyl residue at the R1 side chain enhances the affinity to bone, whereas nitrogen residues at the R2 side chain account for their potency, mechanism of action, and side effects. Developed and traditionally used to soften water in irrigation systems in the 19th century, BPs were introduced into clinical medicine in the 1970s and 1980s in the treatment of Paget’s disease of bone and hypercalcemia of malignancy. Bisphosphonates (BP) may act via many signalling pathways, some of which are specific for a given BP.

First of all two groups of BPs have been identified to date acting in a different ways:

  1. Non-amino bisphosphonates act through ATP block producing toxic analogs of ATP and causing cells death.
  2. Amino bisphosphonates act through inhibition of an enzyme called farnesyl pyrophosphate synthase, an enzyme present in in the 3 hydroxymethyl glutaryl Co A reductase pathway.

Other oossible intracellular pathways have been proposed some of wich are specific for some bisphosphonates. These includes:

- Altering key apoptotic proteins, specifically increasing BAX and decreasing Bcl-2.

- Activating mitochondrial pathway via translocation of apoptosis iducing factor.

- Inhibiting mitochondrial adenine nucleatide translocase (ANT), known to be involved in causing apoptosis

- Inducing ApppI ( an densoine triphosphate analog), which triggers direct apoptosis through blockade of mitochondrial ANT.

- Inhibiting metalloproteinases necessary for proteolytic degradation of the extracellular matrix (ECM).

- Inhibiting cancer cell adhesion ( ICMA-1, VCAM-1) and prevents cancer cells spreading at lower concentration than those required to cause apoptosis.

Main characteristics of BPs are:

- Poor oral bioavailability

- High affinity for, and accumulation in bone

- Target FPP synthase in osteoclasts

- Efficiency across a broad spectrum of osteoclast mediated diseases

Stimulated by the launch of alendronate, the first potent oral aminobisphosphonate, the mechanisms of this drug class were elucidated in the ’90. After parenteral and oral administration in which less than 1% is absorbed, BPs bind to hydroxyapatite crystals and concentrate at skeletal sites where active remodeling takes place. Following embedding into the skeleton, BPs inhibit osteoclasts activity and, under acidic conditions in resorption lacunae, are incorporated into osteoclasts.Nitrogen-containing BPs, the most widely used class of antiosteoporosis drugs, which includes alendronate, risendronate, ibandronate, and zolendronic acid interfere with the mevalonate pathway and inhibit Farnesyl Pyrophosphate synthase (FPPS). FPPS is the enzyme that generates Farnesyl Pyrophosphate and Geranyl-geranyl Pyrophosphate (GGPP), essential for post-translational isoprenylation reaction of small GTPases. These enzymes are able to modulate and coordinate subcellular protein trafficking, cell survival, and cytoskeletal integrity (called Ras, Rho, Rac, Rap).The potency of BPs depends upon the inhibitory effect on FPP synthase activity and the affinity for mineral bone.

In theory, the enzyme hydroxymethy glutaryl (HMG) Coenzyme A reductase inhibitors ( also called “statins”), used usually in therapy for reducing plasma cholesterol, which inhibit the production of mevalonate, also have an osteotropic effect. Due to their lipophilic properties, they preferentially target the liver, but not the skeletal tissue. Inhibition of FPPS by BPs results in decreased osteoclast activity, enhanced osteoclast apoptosis and a profound antiresorptive affect. A relevant finding obtained from bone biopsies of patients treated for long term with bisphosphonates is the increased number of giant, hypernucleated osteoclasts that are detached from bone lacunae and undergone slowly a protracted apoptotic process. (see Manolagas NEJM ). Apart from their specific antiosteoclastic activity, BPs protect osteoblasts and osteocytes against apoptosis, enhancing osteoblastic differentiation, and increasing osteoblastic production of Osteoprotegerin.Since the first BP, alendronate, was approved in 1995 these agents have been the first therapy for treating postmenopausal osteoporosis, male osteoporosis, glucocorticoid-induced osteoporosis, Paget’s disease of bone, Hypercalcemia of malignancy, multiple myeloma of bone, and skeletal metastases.

Bone loss associated with aromatase inhibitor therapy in women with breast cancer, which is associated with very low estrogen levels, has been treated with zolendronic acid administered twice per year.In malignant skeletal diseases, intravenous BPs ( such as zolendronic acid 4 mg, or pamidronate 90 mg) are administered every 4 weeks or also more frequently. The shorter therapy interval is required in order to control excessively enhanced bone resorption in malignant conditions. Under this regimen, the rate of side effects is considerably higher, including renal toxicity and the development of osteonecrosis of the jaw (ONJ), particularly in patients with myeloma or breast cancer following dental procedures. The reported decrease in hip fracture rates in long term clinical studies reported for osteoporosis treatment results from multiple factors. BP effectively reduce fracture risk in postmenopausal women over a period of at least 10 year, but preclinical studies demonstrated that they also negatively affect bone quality.

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Bone quality

Not all fractures have the same pathogenesis or structural abnormalities that cause bone fragility. Some fractures are associated with reduced tissue mineral density; in others, there is a reduced density of osteocytes.Women with fractures may have high, normal, or low rates of remodeling. Some women with fractures have a negative balance in the bone multicellular units owing to reduced bone formation, increased bone resorption, or both; other women with fractures have no negative balance in the bone multicellular unit balance.The heterogeneity of mechanisms suggests that all patients with fragility fractures should not be treated in the same way.In most postmenopausal women, the remodeling rate is high; in other words a large number of bone multicellular units excavate cavities while other units are at various stages involved in the completion of remodeling. When an antiresorptive agents is given, this steady state is perturbed. The birth rate of new bone multicellular units decreases quickly when treatment is started, whereas the many bone multicellular units at various stages in the remodeling cycle complete the remodeling process by depositing a volume of new bone that reduces the depth of the escavated site.The newly deposite bone undergoes primary mineralization during the deposition of osteoid (normally a rapid process) and then slower secondary mineralization with enlargement of newly yet formed crystal occurred thereafter.The increased tissue mineral density and reduced porosity slightly improve bone strength. During treatment with antiresorptive agents, the slow remodeling rate and the reduced depth of a decreased number of excavated sites produces bone loss and structural decay also if more slowly than before, and bone fragility reemerges.Fractures continue but are less frequent than in untreated controls with a rapid remodeling and a negative balance in bone multicellular units exponentially increase bone fragility.

Antiresorptive agents finally slow the progression of fragility by suppressing the rate of remodeling and reducing the depth of resorption in each of the reduced number of bone multicellular units engaged into remodeling bone.Since remodeling is slow during treatment with antiresorptive agents, more time is available for secondary mineralization of new mineral bone both in sites actively resorbing before drug exposure and in sites distant from endosteal surface. So that slower remodeling allows increased bone mineral density with more homogenous distribution of mineral between adjacent regions.

However greater secondary mineralization rate increases tissue stiffness, thereby predisposing to bone microdamage.

Whereas the greater homogeneity in tissue density offers less resistance to the propagation of cracking.

Reducing remodeling may also reduce removal of microdamage in bone.

Studies in dogs shows a nearly 30% decline in material toughness ( the normalized energy to fracture ) over 3 years of reatment at doses that stimulate those used in treating osteoporosis in postmenopausal women. This creates a material more brittle that untreated bone, facilitating microdamage, which, combined with the natural suppression of remodeling to repair it, significantly increases its burden in bone.Micro-damage accumulation is likely a consequence of the increased brittleness and reduced toughness, not its cause.

HR-pQCT

Advances in non invasive techniques are likely to provide insights into the effects of these therapeutic agents on bone structure and increasingly accurate information concerning the structural heterogeneity of bone fragility from patient to patient and so may improve the sensitivity of the prediction of fracture risk.Whereas DEXA has become the most commonly used technique worldwide to predict fracture risk and assess response to therapy, based on a two dimensional interpretation of skeletal tissue; it’s increasingly evident that it provides limited interpretation of three dimensional skeletal properties and so on its structural charateristics.Other imaging modalities such as CT and MRI offer considerable greater charaterization of bone architecture, but their software and technical evolution has not been validated until recently.

High Resolution three dimensional peripheral QuantitativeTomography (HR-pQCT) device has been developed by Xtreme CT, Scanco Medical AG, Bassersdorf, Switzerland in order to provides measures of bone microachitecture and micro Finite Element analysis software for numerical quantification of mechanical properties of bone in vivo.In the following diagram we can see the difference between osteopenic postmenopausal women and osteporotic women with fracture using DXA (BMD) and HR-pQCT parameters. Clearly the differences between two groups is more evident with the later technique.

 


This system, first described in detail by Andreas Laib on 1998, uses a two dimensional detector array in combination with a 0.08 mm point-focus X ray tube, enabling the simultaneous acquisition of a stack of 116 high-resolution parallel CT slices, using an effective energy of 40 keV, X-ray tube current of 95 mA, slice thickness of 89 μm, field of view of 90 mm, image matrix of 1536 x 1536 pixels, and pixel size of 82 μm (voxel size).Older 3-D pQCT devices used before have a voxel size of 165 μm, the need to have an higher resolution value is due to adequately solve the distance between the trabecular ridges ( about 300-500 μm) and not necessary to resolve individular trabeculae (100 μm or less ). At each site 110 CT slices were obtained, thus delivering a three dimensional representation of about 9 mm in the axial direction. The arm of leg of the patient was immobilized during the examination in an anatomically formed carbon fiber shell. An anteroposterior scout view was used to define the measurement region. Briefly, a reference line was manually placed at the endplates of the radius and the tibia. The first CT slice was 9.5 mm and 22.5 mm proximal to the reference line for the distal radius and tibia , respectively. The effective dose was less than 3 microSievert per measurement with a measurement time of 2.8 minutes.Quality control, based on Shewart rules, was monitored by daily scans of a phantom containing rods of HA ( densities of 0, 100, 200, 400, 800 mg HA/cm3 ) embedded in a soft tissue equivalent resin (QRM Moehrendorf, Germany).The entire volume of interest was automatically separated into a cortical and trabecular region using a threshold based algorithm. The threshold used to discrimite cortical from trabecular bone was set to one third of the apparent cortical bone density value (D cort). Mean cortical thicknes (CTh) was defined as the mean cortical volume diveded by the outer boe surface.Trabecular bone density (D trab) in gHA/cm3 was compouted as the average mineral density whitin the trabecular volume of interest.Trabecular bone volume (BV) fraction (BV/TV Trabecular volume %) was then expressed from trabecular density assuming fully mineralized bone to have a mineral density of 1.2 gHA/cm3

BV/TV %= 100 x 1200 mgHA/cm3

Because the thickness of every trabeculae cannot be measured accurately because of partial volume effects, a thickness independent algorithm was used to assess trabecular structure.First, a mid-axis transformation method was used to identify trabecular elements and the distance between them assessed threedimensionally using the distance transform method. Trabeculae cannot be resolved at their correct thickness because of partial volume effects, to avoid this problem the center point of every trabecula is detected in the gray-level image and called the 3-D ridges. Trabecular number is taken as the inverse of the mean spacing of the ridges.Trabecular number (TbN, mm-1) was defined as the inverse of the mean spacing of the mid-axes and is thus truly three-dimensional and it does not depend on “a priori” assumptions regarding the plate or rod-like nature of the underlying nature.

TbN = 1/Mean Tb space = nTb/mm

Trabecular thickness (TbTh, μm) and separation (TbSp, μm) were derived from BV/TV and TbN using a standard methods from histomorphometry.

TbTh = (BV/TV)/TbN

TbSp = (1-BV/TV)/TbN

Distance transformation techniques also enable the calculation of intra-individual distribution of separation (TbSp SD, μm) quantified bt the umeber of Standard Deviation (SD) of the separation mean, a parameter reflecting the heterogeneity of trabecular network.For follow-up measurements, an algorithm automatically uses the cross-sectional area (CSA, mm2 ) within the periosteal boundary of the radius and tibia to match the volumes of interest (VOI) on the baseline and fllow-up scans, and thus only the bone volume common to previous scans is used to assess density and microarchitectural measurements.Thus, of the initial 110 slices, on average 103 (range 93 to 108) were analyzed in the follow-up scans.

The outcome variables used in this analyses included volumetrical bone density (gHA/cm3) for entire (Dtot), trabecular (Dtrab), and cortical (Dcort) regions; cortical thickness (CTh, μm), trabecular bone volume fraction (BV/TV, %), trabecular thickness (TbTh, μm), trabecular number (TbN*, mm-1), trabecular separation (TbSp, μm), and intra-individual distribution of separation (TbSp SD, μm).

Interestingly trabecular and cortical densities obtained with HR-pQCT are only moderately related to each other and trabecular density is strongly correlated to trabecular achitectural measurements at both distal radius and tibia. On the contrary, cortical density is higly correlated with cortical thickness but weakly correlated with trabecular architecture in normal subject.Postmenopausal osteopenic and osteoporotic women show density and architectural parameters significantly different, with the exception of cortical density. Compared with those classified as osteopenic, osteoporotic women have lower bone density, decreased trabecular number and trabecular thickness, increased trabecular separartion and intra-individual distribution of separation, and decreased cortical thickness. Osteopenic women with and without an history of fracture did not differ with regard to BMD (measured with classic Hologic densitometr) at lumbar spine and femoral neck, nor in HR-pQCT measurements at the distal tibia. However, at the distal radius density and architectural parameters were significantly different in women with an history of fracture compared with those with no previous fractures.In men trabecular bone volume declines similarly as women over life, however the microstructural basis for the decrease in trabecular volume differ between sexes. In women there appear to be loss of trabeculae with decrease in trabecular number and incresed intertrabecular space, whereas in men the primary mechanism for the decrease in trabecular volume is trabecular thinning.

This mechanism in turn is likely to have a significant impact on age related changes in bone strength in women compared to men, because the reduction in trabecular number has a 2 or 5 times greater impact on bone strength compared with reduction in trabecular thickness that result in similar decreases in bone volume.

Micro-finite element analysis using HR-pQCT

Micro-finite element analysis (μFE) tecniques applied to HR-pQCT data sets provide an estimate of bone mechanical competence (stiffness) that distinguishes between groups of subjects with and without fractures.Each subvolume of HR-pQCT image oof the distal radius and distal tibia is converted to a micro-finite element (μFE) with an element size of 82 x 82 x 82 μm3.The HR-pQCT measurement, as writed above, include 116 slices, corresponding to a 9.02 mm sections along the axial skeleton, with a nominal voxel size of 82 μm.The mineralized phase was thresholded automatically, using Laplace-Hamming filter followed by a global threshold using a fixed value of 40% of the maximal grayscale value of the images.Using customized element-by-element pre-conditioned conjugate gradient solver, 6 μFEs were performed for each model, representing 3 uniaxial compression tests along 3 imaging axes and 3 uniaxial shear tests.The trabecular bone tissue is considered as an isotropic, linear elastic material with a Young’s modulus (E) of 15 Gpa and a Poisson’s ratio of 0.3 for all uniaxial model.The general anysotropic stifness of bone matrix is transformed into a new value through the calculation by means of appropriate algorithm, called Powell’s method, of full orthotopic stiffness tensor value by best orthotopic symmetry through the new chosed coordinate system formed by chosed 3 orthotopic axes ( X1, X2, X3 ) representing the best orthotopic symmetry calculated using an optimization procedure.The elastic constants and stiffness matrix moduli were sorted so that E11 was in the medial-to-lateral direction ( representing the lowest axial modulus), E22 along the antero-posterior direction, whereas E33 was in the direction of the highest axial direction.

Finally 6 elastic moduli were derived from the orthotopic system tensor value:

3 Young moduli: E11 < E22 < E33 for unaxial compression tests

3 Shear moduli: G23 < G31 < G12 for unaxial shear tests

Several studies have reported that HR-pQCT parameters discriminate between postmenopausal women with and without fractures, whereas BMD by DEXA did not.Melton LJ III and Delmas PJ groups reported that decreased vBMD, microstructure, and stiffness estimated by μFE of the radius are associted with forearm fracture in postmenopausal women.Patients studied with postmenopausal osteopenia, radius but not tibia HR-pQCT measurements discriminated between those with and without fractures. It is also important because tibia is a weight bearing bone and it would be predicted that mechanical loading would result in a relative sparing at this site. The study by Cohen A. confirms these data by providing the evidence of cortical and trabecular microarchitectural deterioration at both radius and tibia in premenopausal women with idiopatic osteoporosis, whether or not they have had fracture.Estimated stiffness was significantly lower in all directions at both radius and tibia. Noteworthy was the finding that trabecular bone microachitecture and stiffness were severely affected at radial site in women with low BMD (measured with standard Hologic densitometry) who had an adult low trauma fracture.

Bisphosphonates

The excessive suppression of bone remodeling by high doses of bisphosphonates is thought to compromise bone integrity by accumulation of microdamage (microcraks).However,the microdame ge accumulation has been demonstrated to peak during early period of high dose bisphosphonate treatment and the drugs does not continue to accumulate with longer treatment periods, Determinats of bone strength including ultimate load, stiffness, anergy to failure as well’s other material properties including bone maximum stress and modulus have been shown to be unaffected and preserved after three years of daily alendronate treatment also in preclinical animal models.The role of other material properties has been found to be altered by bisphosphonate treatment but they role in alteration of fracture stiffness is less evident. We talk mainly of alterations of bone mineralization quality, collagen ultrastructural quality, and mineral hydroxyapatite quality. Concerning the possible role of increased quantity of mineralized bone it is quite clear that higher bone mineralization is beneficial in increasing bone stiffness and reducing the incidence of new fractures at any site. The report of possible brittleness of new bone formed and the increasing report of new subtrochanteric and mid shaft femur fractures have not be considered osteoporotic fractures, so not related to disease treated by bisphosphonates, but related to intensity work load in a possible normal bone.

The change in bone tissue is more likely caused by larger accumulation of advanced glycation end-products, called AGEs and directly related to increased glucose levels.AGEs are the by-products of the formation of collagen cross-links by non-enzymatic processes, and naturally accumulates in bone as it ages. Undernormal bone turnover rates, AGEs are prevented from accumulating to high levels. When bone turnover is suppressed, however, they can accumulate, and laboratory studies show them to be associated with increased brittleness.The micro-damage accumulation, and possibly the build-up of AGEs in the bone extracellular matrix, can only be reversed by bone anabolic agents such as teriparatide.

Intravenous BPs and in particular zolendronic acid may be associated with hypocalcemia, renal toxicity, and an acute phase reaction with flulike symptoms during drugs infusion. The latter is thought to be due to extraskeletal effects of aminobisphosphonates, the release of cytokines from macrophages, and the activation of T lymphocytes linking to γδ T cell receptor (see NEJM letter).Accordingly, aminobisphosphonats, and in particular zolendronic acid, may also induce apoptosis in breast cancer cells, although the clinical relevance of this effect is not clear.We know that bone remodelling is a process involving T lymphocytes, bone marrow stromal cells, machrophages (antigen presenting cells) in a complex signalling pathway involving the activation of osteocytes, osteoblasts and finally osteoclasts through a signaling sequences very complexes and those more intensely studied require TNF alfa related factors and their receptors (RANK/RANKL/OPG). So that in any changes of bone turnover level a true inflammatory-like pathway is activated at bone marrow-trabecular interfaces.

According to my opinion all side-effects founded in long term studies using bisphosphonates and in particular after their parenteral administration one a months or yearly is due to an increase in local inflammatory tissutal answer, finally accounting for:

  1. Osteonecrosis of the jaw (ONJ)
  2. Atrial Fibrillation (AF)
  3. Esophageal cancer
  4. Musculoskeletal pain
  5. Atypical fractures due to increased skeletal fragility at diaphyseal or subtrochanteric femur regions.

Since late 2003 there have been reports in the literature of a possible association between bisphosphonate use and the appearance of avascular necrosis of the jaw. Marx on 2003 described a group of 36 American patients who received either pamidronate or zoledronate iv for the management of bone disease associated with metastatic cancer, multiple myeloma and osteoporosis and who subsequently developed avascular necrosis of the jaws. In the majority of patients, the latter condition developed after dental extraction, but in about 30% of cases, it apparently occurred spontaneously.

Bisphosphonates related osteomyelitis (BON) and necrosis of the jaw possibly results from the inability of hypodynamic and hypovascular bone to meet an increased demand for repair and remodeling owing to physiological stress (mastication), iatrogenic trauma ( tooth extraction or denture induced local injury ), or tooth infection in a environment that is both trauma intense and plenty of bacteria.Cofactors may include the use of other medications with antiangiogenic properties such as glucocorticoids, diabetes mellitus, irradiation of jaw bone, peripheral vascular disease, hyperviscosity syndrome such as in multiple myeloma.Bisphosphonate related osteomyelitis (BON) is a true bone infection due to direct effect of bisphosphonates on bone turnover and subsequent physiolgical reaction even more increased if we look at region with increased work load and stress such as daily work activity we spend during mastication. A great work load per cm square is exerted on oral cavity bones so that these bone regions require a very intense answer by extracellular matrix structures.

Concerning the presence of Advanced glycation end products, we know that increasing the concentration of glucose, the link of glucose to all proteins present in our body increases from hemoglobin to proteins present in the ocular structures (both cornea and retinal epithelial cells). Glycation reactions changes the biochemical properties of enzymatic proteins, or receptor proteins, or structural proteins such as collagen fibers. We can postulate that also the presence of increased Atrial Fibrillation should be attributed to altered glycations and expression at myocardial level of proteins forming ion channels, and in the presence of altered calcium homeostasis ( as we have during osteoporotic bone resorption) we have an increased probability to develop myocardial depolarizarion leading finally to Atrial Fibrillation.Cellular electrophysiolgical studies have revealed a marked reduction in the densities of L-type volatage gated Calcium channels, transient outward Potassium currents, and ultrarapid delayed rectifier Potassium currents in atrial myocites of patients affected by Atrial Fibrillation.Interestingly similar ( but not identical ) changes are present in canine models of Atrial Fibrillation, where changes in ions currents are correlated with reduced expression of the underlying channels forming subunits.In both human and canine Atrial Fibrillation, reduced Calcium voltage currents seem to be enought to explaine the reduction action potentials in duration and effective refractory period characteristics of remodelling atria.In addition the sarcoplasmic expression of Calcium dependent ATPase is reduced in myocites, suggesting that calcium cycling is affected in atrial fibrillated myocites.

References

Seeman E, Delmas PD. Bone quality – The material and structural basis of bone strength and fragility. N Engl J Med 2006;354:2250-61.

Watts NB, Diab DL. Long term use of bisphosphonates in osteoporosis. J Clin Endocrinol Metab 2010;95:1555-65.

Liberman UA, Weiss SR, Bröll J et al. Effects of oral alendronate on bone mineral density and the incidence of fractures in postmenopausal osteoporosis. The Alendronate Phase III Osteoporosis Treatment Study Group. N Engl J Med 1995;333:1437-43.

Orwoll E, Ettinger M, Weiss S et al. Alendronate for the treatment of osteoporosis in men. N Engl J Med 2000;343:604-10.

Reid IR, Brown JP, Burkhardt P et al. Intravenous Zolendronic acid in postmenopausal women with low bone mineral density. N Engl J Med 2002;346:653-61.

Bone HG, Hosking D, Devogelaer JP et al. Ten years experience with alendronate for osteoporosis in postmenopausal women. N Engl J Med 2004;350:1189-99.

Black DM, Delmas PD, Eastell R et al. for the HORIZON Pivotal Fracture Trial. Once-yearly zolendronic acid for treatment of postmenopausal osteoporosis. N Engl J Med 2007;356:1809-22.

Cummings SR, Schwartz AV, Black DM. Alendronate and atrial fibrillation. N Engl J Med 2007;356:1895-6.

Lyles KW, Colòn-Emeric CS, Magaziner JS et al. HORIZON Recurrent fracture trial. Zolendronic acid and clinical fractures and mortality after hip fracture. N Engl J Med 2007;357:1799-809.

Lenart BA, Lorich DG, Lane JM. Atypical fractures of the femoral diaphysis in postmenopausal women taking alendronate. N Engl J Med 2008;358:1304-6.

Weinstein RS, Roberson PK, Manolagas SC. Giant osteoclast formation and long term oral bisphosphonate therapy. N Engl J Med 2009;360:53-62.

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Wednesday, April 14, 2010

FGFs : New player in bone metabolism.

                  

   
   

Rickets

Old English term for Rickets was “Wrickken“ as described by Glisson F, Bate G and Regemorter A on 1651 as a common disease present in children. Near 350 years have been elapsed since the first monograph publication on wrickken and many spectacular advances in our understanding of Vitamin D dependent metabolism have enriched our knowledge wallet.

Last century was plenty of relevant discoveries from isolation of nuclear receptor for Vitamin D, identification of metabolic pathways of vitamin D synthesis, activation and degradation, until to isolation of genes coding for enzymes and co-factors as well’s hormonal factors involved into vitamin D cycle.

We can define rickets the defect in bone mineralization leading to formation of a normal bone matrix whithout deposition of calcium salts. Anatomically speaking this alteration in bone structure is called “osteomalacia”, whereas clinically propension to multiple fractures, short stature, bone deformities and renal alterations are often found. Initially the defect was ascribed to a loss of vitamin D expecially in young adults presenting lower exposure to sunlight; later it was clear that different forms of rickets are present; some involving abnormalities in vitamin D metabolism, other involving renal cells alterations.

It’s quite surprising that the horthologues of oncogenes designed such as “Interruptor-1” and “Interruptor-2” have been demonstrated to be involved into phosphate homeostasis. Int-1 is Wnt family growth factors, whereas Int-2 is FGFs family growth factor; both are very important in molecular embriology influencing cellular condensation and diffusion. Phosphate ions with carbon and azote ions, can be considered the base for live organisms in the earth, forming the backbone of all biomolecular structures. It is surprising that a so higly conserved and sophysticated biochemical pathway has been created to conserve adequate levels of phosphate in our organisms.

Interestingly, it is not at present clear the relation between bone loss and kidney stone formation. Excessive bone reabsorption clearly leads to hypercalciuria and hyperphsophaturia for example by excessive production of parathyroid hormone or vitamin D3. Excessive increase in urinary calcium excretion from bone can also be observed in osteogenesis imperfecta and in McCune-Albright syndrome; however, nephrolithiasis is very rare in these disorders.

On the other side, many studies have reported lower bone density in renal calcium stone formers compared to controls. The mechnisms underlying these findings is not clear, but may involve hyper responsiveness to calcitriol or to parathormone or bone abnormalities. However, low bone density in calcum stome formers has not been associated with increased bone resorption or with any gene mutations or polymorphisms.

According to my opinion, whereas osteoporotic bone disease can be truly considered an alteration of bone cells, osteomalacia (rickets) is better considered a bone symptom of a kidney disease. In the first situation we have altered bone turnover due to alterations of bone regulatory factors, in the second we have bone disease as secondary and sporadic manifestation of primary kidney disease.

Among the causes of defects of inadequate mineralization of bone (osteomalacia) and defective mineralization of cartilage (rickets) are renal phosphate wasting disorders that produce hypophosphatemia.

Kidney stones

Urinary PH determines the solubility of various substances in urine, Low PH decreases uric acid solubility but prevents calcium-phosphate crystal formation in contrast to a PH > 7 that augments urate solubility but precipitates calcium-phosphate salts. Urinary PH depends on the proton load in the diet and on the ability of kidney to buffer free proton in the urine. Renal acidosis is due to a defect of the renal tubule in secreting protons while buffers are normally produced, resulting in urinary PH > 5.5 and in metabolic acidosis. Patients with renal acidosis frequently have hypercalciuria and hyperphosphaturia. This may be due to calcium and phosphate release from bone because of proton buffering by bone.

Nephrolithiasis and nephrocalcinosis are frequent in these disorders. Distal renal acidosis is due to mutations in the chloride-bicarbonate exchanger or in proton ATP ase subunits.

Uric acid

Uric acid stones are less frequent than calcium stones and they represent 5 to 10% of nephrolithiasis. Uric acid is the final breakdown product of purine metabolism and it also derives from amino acid catabolism. Two-third of uric acid production are eliminated by the kidneys in humans. Uric acid is filtered at the glomerulus and then it is almost completely reabsorbed in the initial part of the proximal tubule by at least two transporters, called URAT1 , coded by gene SLC22A12) and GLUT9 ( coded by gene SLC2A9). A member of the ATP binding cassette family (ABCG2) is expressed in proximal tubular cells and secretes urate into the urine.

Two factors increase the risk of uric acid stone formation: low PH and hyperuricosuria. Genetic disorders can increase uric acid production or alter urate tubular transport. An increase in uric acid production induces hyperuricemia, as in the case of hypoxantine-guanine- phosphorybosyl transferase or in glucose-6-phosphatase deficiencies, and in phosporybosyl pyrophosphatase synthetase over-activity.

In contrast mutations in urate transporters result in hypouricemia and hyperuricosuria. Hence loss-of-function mutations in the URAT1 transporter decrease urate reabsorption in the proximal tubule. Functional experiments demonstrated that SLC9A2, is expressed at the apical and basolateral sides of proximal tubular cells, it transport urate, and that polymorphisms decrease urate reuptake increasing uric acid secretion in urine.

Inactivating mutations in tha ABCG2 gene have been identified as cause of gout increasing uric acid concentration in plasma.

Oxalate

Elevatd urinary oxalate excretion is critical for the growth of renal stones, Oxalate comes from the diet and it is produced by the liver and arythrocytes from glyoxalate. It is filtered freely at the glomerulus and probably reabsorbed ad then secreted in the proximal tubule. In the intestine, oxalate is also absorbed and secreted, but absorption exceeds secretion.

Enzymatic defects (primary hyperoxalurias) can induce oxalate overproduction.

The gene SLCA26A6 encodes an oxalate-chloride exchanger that is expressed in the intestine and in the renal proximal tubule. The disruption of this gene in mice results in an increase in oxalate plasma concentration, hyperoxaluria and renal calcium oxalate stone formation. The role of SLC26A6 is probably to secrete oxalate in feces. Its role in proximal tubule is not clear. Mutations in this gene have not been identified in humans.

Claudins

The selecive permeability of the intercellular unctions to calcium and magnesium ions is due to expression of claudin-16 , also known such as paracellin. This protein acts as a specific gate for clacium and magnesium. Mutations in claudin 16 abolished the permeability of the intercellular pathway to calcium, and are a rare cause of hypercalciuria, hypomagnesemia and calcium renal stones.

Variants of the gne enchoding claudin 14 have recently been asociated with kidney stones and low bone mineral density in a genome wide association study performed in patients from the Netherlands, Iceland, and Denmark, Claudin 14 is a tight junction protein expressed in the proximal tubule and in the loop of Henle. The mechanism by which these variants are associated with renal stones is unclear since they were not associated with calcium or phosphate concentration in plasma or urine, but only associted with serum PTH and bone markers.

OSTEOMALACIA

Vitamin D metabolism abnormalities

Rickets-Vitamin-D-dependent-type-I 1ahydroxylaseD3 (Ch12q14)

Rickets-Vitamin-D-dependent-type-II              Vit.-D receptor (Ch12q13-14)

Rickets-PseudoVitamin-D-deficiency              1ahydroxylaseD3(Ch12q13.3) point mutation

Kidney Proximal tubular defect

Rickets hypophosphatemic with Hypercalciuria       NTP2c gene (HHRH)(NPT2 genes Ch5q35.1-q35.3)

Autosomal dominant hypophosphatemic Rickets    FGF23 gene (ADHR) OMIM 193100 of the young (Ch.12p13.3)

X Linked Hypophosphatemic Richets                       PHEX gene (HYP) like OMIM adult female (Ch. Xq22.1)

Tumor Induced Osteomalacia                                 FRP4 gene(OHO) (Ch.7q14.1)

Hyperphosphatemic Familial Tumoral Calcinosis          KLOTHO (HFTC) OMIM 21900

Hypeostosis-hyperphosphatemia syndrome                  GALNT3 (HHS) OMIM 610233

Renal Tubular Acidosis type II                              NPT2a gene (Fanconi’s Syndrome) (Ch5q35.1-q35.3)

 

Distal tubular defect

Renal-Tubular-Acidosis-type-I          Basolateral- Cl/HCO3   (RTA Distal)                                               Proton-ATPase

Hyperkalemic RTA type IV    Hyporeninemic Hyperaldosteronism

Bartter’s syndrome              Na-K-2Cl transporter mutation

(Henle loop hyperca)           K channel-calcium sensing receptor

                                       Chloride channel (CICKa-b)-Barttrin

Gitelman’s syndrome          Na-Cl transporter thiazide sensitive

(distal hyperca)

Dent’s disease                   Voltage gated Chloride channel

INHERITED BONE DISEASES

Autosomal dominant Aplasia of                             FGF10 gene

Lacrimal and Salivary glands

Autosomal dominant Cerebellar                             FGF14 gene

Ataxia

Craniosynostosis disorders                  FGFR type 2 (Pro250X)

Achondroplasia                                        FGFR type 3 (Ch4p16.3)

Hypochondroplasia                                  FGFR type 3

Spondyloepiphyseal dysplasia *          FGFR type 3 stop codon

Stickler syndrome *                            FGFR type 3 Lys650Glu

*thanatophoric dysplasia type I and type II.

Pfeiffer syndrome                              FGFR type 1 Pro252Arg

Apert syndrome                                 FGFR type 2 Pro253Arg

Muenke craniosynostosis                   FGFR type 3 Pro 250Arg

Crouzon syndrome                            FGFR type 2 Cys342Arg

Jackson-Weiss syndrome                   FGFR type 2 Cys342Arg

Phosphate wasting is either inherited as X-linked hypophosphatemic rickets or autosomal dominant hypophosphatemic rickets, or acquired, as can occur in patients with a variety of benign mesenchimal tumors such as hemangiopericytomas, fibromas, angiosarcomas. Osteomalacia induced by tumors is invariably curable if the tumor can be found and resected, indicating that it may have an humoral basis. However the pathogenesis of rachitic syndromes requires also defective mineralization coupled with phosphate wasting.

Phosphatonins have been demonstrated to be able to impair the action of kidney 1a hydroxylasis, activating 24-hydroxylasis, and mediating the parathyroid action on many cell types, including kidney proximal cells.

However no clear action at bone forming units has been at present demonstrated by “phosphatonins” per se.

NPT2 and NHERF

We are understanding the mechanisms exerted by epithelial scaffold proteins in regulation of renal phosphate handling. These PDZ-containing proteins are able to form macromolecular complexes with true Na/Pi channels.

Called Na+/H+ Exchangers Regulatory Factor (NHERFs), they are known to be present on apical microvillar structure (NHERF1) or at the base of microvilli in the vescicle rich domains (NHERF2). They are ancillary cytoplasmic proteins, responsive to hormonal stimulation such as parathyroid hormone, and directing the localization of ion channel proteins (NPT2) at specific sites of cellular membrane. Their action has been demonstrated for microvillar apical membrane of proximal kidney tubular cells, but they probably, as PDZ-containing proteins, are ubiquitariously active in regulating the activity, internalization and recycling, of true receptors.

Their action is strictly dependent from transmembrane potential and finally from different concentration of sodium and potassium ions outside and inside cells respectively. As we know cell life is linked to the presence of this different concentration, allowing the formation of true concentration gradient across a lipid bilayer.

Moreover they are co-responsive of the presence of a difference in proton concentration (i.e.PH) across plasmamembrane. The PH difference across lipid bilayer between inner and extracellular fluid account for a different solubility or organic and inorganic salts as well’s of cathalization of enzymatic reactions possible only at a given PH.

Finally, these apparent unuseful PDZ-containing proteins should give the life to an inhert lipid bilayer; making it responsive to extracellular hormonal signals and so orchestrating the action of an uncohordinated lipid structure.

Their presence at specific sites of plasma membrane explaines the localization and activity of the products of SCL34A1 and SCL34A3 genes located on chromosome 5q35.1-35.3 and coding for (NPT2a) Na/Pi exchanger type IIa and (NPT2c) type IIc respectively. The sodium-phosphate cotransporter NTP-2c is responsible for the bulk of phosphate reabsorption in proximal renal tubules and its alteration is the cause of hypophosphatemic rickets with hypercalciuria. The putative “phosphatonin” should directly inhibit renal sodium-phosphate cotransporter.NPT2c is the primary hormone – regulated renal phosphate transporter, localized at the apical membrane of cells of proximal tubule in each nephron. It accounts for 80% of sodium dependent phosphate reabsorption.Interestingly dietary phosphate load causes a significant down-regulation of NPT-2c.

A distinct apical membrane sodium-phosphate cotransporter called NTP-2a is present in nephrons, sharing homologies with the former responsible for Proximal Renal Tubular Acidosis also called Fanconi’s Syndrome.It is quite consequential that the phosphaturic action exerted by FGF23 at kidney level is done by a down-regulation or block of the action of NTP-2 gene products mediating the reabsorption of phosphate ions.

Hyp mice experiments, showing a 50% reduction in NPT2, and an increase in FGF23 can be a perfect example of what happen in phosphate wasting syndromes.

FGF23

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A paper of Shimada et al. on PNAS (2001) identifies a member of the fibroblast growth factor family, FGF23, as the humoral factor that is secreted by tumors to cause tumor-induced osteomalacia. They cloned a cDNA from a hemangiopericytoma that caused hypophosphatemic osteomalacia and found clones identical to FGF23, which was recently identified by positional cloning as the gene responsible for autosomal dominant hypophosphatemic rickets. When injected into mice the recombinant FGF23 produces mild phosphaturia and hypophosphatemia; interestingly Chinese Hamster Ovary (CHO) cells - FGF23, when grown as tumor in nude mice, fully reproduced the human syndrome of severe hypophosphatemia, growth retardation and rickets in the growth plates, deformities in the skeleton, reduced mineralized matrix and seems of unmineralized osteoid in bone. FGF 23 was expressed at high levels in the tumor from which it was cloned, and as recently reported by another group it is also expressed at high levels in other tumors associated with acquired osteomalacia.

Its expression in bone reach the highest level, in regions of active bone formation, a strong hybridization signal can be seen in osteoblasts lining bone surfaces. Newly formed osteocytes and osteoprogenitor cells are also labeled. In other tissues it has been detected in particular in parathyroid, thymus, brain, heart, and vascular system. If in the past some contraddictory results have been obtained it was due to difficulties in metods of measurements. In particular it is necessary to evaluate if we have to measure the entire FGF23 or only its biologically active portion, its C terminal (Ct) part.

With a 72 aa Ct domain not shared with other family members FGF23 is the largest member of the FGF family. Insight into its functions are provided by demostration that mutations caused hypophosphatemic rickets. Moreover it may seems more soluble that other family members lacking the heparing-binding motif presented in other FGFs.

Whyte KE demonstrated that four unrelated families had a missense mutations in one or two closely spaced arginine residues at position 176 and 179 that cosegregates with rickets, with two families sharing the same mutation. This clustering of missense mutations is a disorder with a dominant inheritance and strongly suggest a “gain of function mutation”.

Shimada et al demonstrated that arginine 179 and S180 is a processing site in FGF23, because they found in CHO cells expressing FGF23, in addition to the mature protein a fragmented protein beginning with S180. It is believed that mutations of the flanking arginine at position 179 could confer a gain of function on FGF23 by blocking its degradation at cleavage site between the unique Ct domain and the FGF-homologue regions.

FGF23 may be the long-sought “phosphatonin”, the phosphaturic factor normally accounting for phosphate homeostasis, independently from parathyroid hormone, so independently from calcium levels. It has been postulated that the main help to FGF23 secretion is the product between the concentration of Calcium and Phosphorus. So acquiring a relevance in regulation of ectopic calcifications such as those present in vascular system with aging.It may be that FGF23 is also secreted by one or more normal tissues as a phosphate regulating hormone, and that the blood level of FGF23 in blood is determined in part by the rate of cleavage by Subtilisin-like proprotein convertase (SPC) at R179/S180. Two fragments are present an entire protein of 32 kDa and C terminal segment of 12 kDa.On 2004 it was demonstrated the complete action of FGF23 on NTP2 renal cotransporter, giving the final answer to identification of FGF23 such as phosphatonin.

Quite recently a longitudinal study demonstrated that in dyalitic patients measurements of FGF23 plasma levels can be useful in assessing normo-phosphatemic patients who should benefits of therapeutic strategies devoted to manage phosphorus balance, considering that hyperphosphatemic hemodyalitic patients show an increased risk of death. This study suggests that hyperphosphatemia in these patients is only partially assessment of risk associated with abnormal phosphorus metabolism. However, measurement of FGF23 could represent a new biomarker in assessing the risk of death in patients with early kidney disease (Wolf M. NEJM August 7, 2008).


PHEX

The other piece of the hypophosphatemic puzzle is the X-linked hypophosphatemic rickets, the most common disorder of renal phosphate transporter.

PHEX belongs to the M13 family of MA clan of Zn-metalloendopeptidases. The prototypic member of this group of type of integral membrane glycoproteins is Neutral Endopeptidase (NEP). These proteins have a short cytoplasmic N-terminal region, a single transmembrane domain, and a large extracellular C-terminal domain with a zinc binding motif.

Other members of this group include:

. Endothelin Converting Enzyme-1 (ECE-1alfa, ECE-1beta and ECE-2)

. ECE-like enzyme/distress induced neuronal endopeptidase (ECEL1/DINE)

. Soluble Endopeptidase/NEP-like enzyme-1/Neprilysin-2 (NL1/NEP2)

. Membrane Matallo Endopeptidase-like 2 (MMEL-2)

. Kell Blood Group Protein antigen (KELL)

Neprilysin is aslo calle common acute lymphoblastic leukemia antigen (CALLA), or CD10, NEP, or Enkephilinase.

The M13 zinc metallo endopeptidases are integrally involved in several essential elements of cellular regulation and physiology as well’s in diseases including renal function defects, bone mineral loss disorder, cardiovascular diseases, arthritis nd inflammatory disorders.In particular PHEX gene is similar to those of NEP family in several important aspects: numebrs of small exons (22 exons characterized for PHEX with a sequence of 749 aminoacids), higly conserved aminoacid Zinc binding motif (HEFTH fof PHEX, HEITH for NEP).

Informations concerning the structure and nature of the PHEX gene product catalytic site was acquired from the analysis of 99 families affected by X Linked Hypophosphatemic Richets (HYP) and compter generated physichemical data and site-directed mutagenesis studies published for M13 and M3 metallo peptidases.

Interestingly full lenght FGF23 and MEFE do not appear to be PHEX substrates. Remarkably PHEX protects full-leght MEPE from proteolysis, notably by catepsin B cleavage in vitro. In addition, osteocalcin is not degraded by PHEX and inhibits PHEX cleavage of PTHrP.

PTHrP is one of the very few naturally occurring substrates cleaved by PHEX.

Osteocalcin is not cleaved by PHEX, the negatively charged Gla residues in osteocalcin are thought to interact with higly conserved charged present in PHEX. Similar charged region is present in MEPE and ASARM proteins.

In the intact MEPE and PHEX may be associated through the interaction with the MEPE C-terminal ASARM motif. This interaction may not necessarily lead to proteolysis. MEPE-PHEX interaction may therefore prevent proteolytic cleavage and release of ASARM peptide by protecting MEPE from localized matrix proteases. PHEX is localized on plasma membrane surface of osteoblasts, with its extracellular long C terminal region ideally situated in extracellular matrix for protein-protein interactions.

Several PHEX mutations has been detected in patients affected by X Linked Hypophosphatemic Richets (HYP) results in sequestration of disease causing PHEX in endoplasmic reticulum and subsequent failure to targeting to plasmamembrane.

PHEX play a major role in mineralization and it is expressed predominantly in bones and teeth.

The bone expression is localized into osteoblasts, osteocytes (not pre-osteoblasts); in teeth it is present in odontoblasts.

Interestingly loss of function of PHEX results in a defective mineralization. Its action on kidney is expressed modulating renal phosphate handling but not directly, suggesting a secondary involvement in regulation of a circulating systemic factor.

Finally it is reasonable to speculate that PHEX may well function as a small peptide protease and also as a matix-protein ligand.

SPC Subtilisin-like proprotein convertase

The SPC are a family of serine proteases, involved in processing of a wide variety of polypeptides including neuropeptides, growth factors, receptors, blood coagulation factors. Their substrates are cleaved at C terminal side where a specific sequence is present . SPC are present at Golgi apparatus and at trans Golgi network, where they act also on FGF23.

Matrix Extracellular phosphoglycoprotein (MEPE)

MEPE was first cloned from a tumor reseacted from a patients with OHO. It belong to a family of proteins that ahve recently been named Shorth Integrin Binding Ligand Interacting Glycoprotein (SIBLINGs). Between them we have:

. Osteopontin

. Matrix extracellular phosphoprotein (MEPE)

. Dentin Matrix Protein 1

. Bone Sialoprotein

. Dentin Sialo Phosphoprotein

. Enamelin

All mapping on chromosome 4q21 and sharing many properties. All these proteins have a links with bone/dentin mineralization and phosphate/calcium salts.

The structure of these proteins contains RGD motif tipical of integrin ligands, glycosylation pattern very similar between them, phosphorylation pattern again similar, and a so called ASARM motif.

ASRM (Acidic Serine Aspartate Rich MEPE associated motif) described a region of these protein able to block the mineralization. However if it is bounded to other extracellular matrix components it may be required a a nucleator of mineralization itself. The ASARM peptide is very stable and it is resistant to know proteases. Free ASARM peptides may also contribute to inhibition of renal phosphate uptake. This mechanism of action is likely to be steric and exacerbating the effect of NTP2 exchanger protein probably with the aids of FGF23.

The normal action of MEPE is to act such as mineralization inhibitor, due to the presence of ASARM fragment normally released from the entire protein by the cathepsin C cleavage.

Another action of MEPE is a dose dependent inhibition of BMP-2 menediated mineralization in a murine osteoblast cell line in vitro, another effect linked to ASARM presence.

Mice models

From studies in hypophosphatemic mice called “hyp mice” and “gyro mice” it was identified the cause of reduced phosphate reabsorption in a gene located in mouse chromosome X coding for PHEX a metalloproteinase enzyme. This proteins was defective in these mice; in hyp mouse the defect was localized primary in kidney, whereas in gyro mouse the defect was located also in the inner ear and so clinically associated with circling behaviour.

KO mice for Na-Pi cotransporter gene called Npt2, showed Pi renal wasting comparable to inactivation of PHEX gene, but in Npt2 KO mice calcitriol responds appropriately to hypophosphatemic challenges, intestinal absorption of both Pi and Ca ensues, and rickets and osteomalcia are absent.

MEPE KO mice have increased bone mass, resistance to aging associated trabecular bone loss, increased mineralization apposition rate and a dramatically accelerated mineralization rate in ex vivo osteoblasts cultures.

Interestingly Vitamin D3 Receptor KO mice have markedly increased levels of mRNA for MEPE expression.

Comparison between these mice models illustrates that renal phosphate wasting can be dissocated from defective synthesis of calcitriol, implying that phosphatonins have at least two independent actions:

1. they inhibit the Pi reabsorption

2. they impair the synthesis of calcitriol

The different phenotypes in Npt2 KO mice and PHEX KO mice also raise the possibility that FGF23 has a direct affect on bone and cartilage that contribute, along with hypophasphatemia to a defect in mineralization.

HYP mice model hepl us in understanding a possible explanation; the HYP mouse model of PHEX inactivation responds to phosphate deprivation, with continued phosphaturia relative to wilde type mice.

Normally phosphate can be cleared from urine by low dietary Pi intake, protecting against Pi depletion. So that decreased FGF23 secretion could be in this view the humoral factor of this response, coupling with at yet unidentifed phosphate sensor, possibly in the intestinal mucosa, to regulate intestinal phosphate reabsorption. Moreover FGF23 may explaine only a local paracrine regulatory role perhaps even unrelated to Pi homeostasis, and only when it is inappropriately secreted into blood may exert a Pi wasting action.

In this scenario, the phosphate wasting in tumor induced osteomalacia would be analogous to the Pi wasting that occurs when tumors overexpress the PTH-related peptides, PTH-rP. PTH-rP is normally a local regulator of cell differentiation, but when overproduction gives it access to the circulation, it co-opts the PTH receptor in kidney to cause phosphaturia.

Conerning FGF23 it is the first FGFs for which mutations are associated with a disease, and althought the othr 22 FGFs share only 4 known receptors, it is likely that FGF23 has a different receptor, because cleavage of its unique C terminal domain inactivates it.

Tumor Induced Osteomalacia

In literature about 70 cases have been described with such rare form of hypophosphaturia, which occurs in association with coexisting tumor and resolve after its excision; with a possible relapsing episodes.

Hypophosphatemia is probably due to a diminished renal phosphate reabsorption and this phenomenon causes a decrease in 1a hydroxylation of vitamin D3.

Tumor are generally of mesenchymal origin ( such as hemangiopericytomas), but also prostate and breast cancer have been described. They are often small and difficult to locate. From Cai Q data on 1994 it was described a “unidentified soluble factor” heat labile that is devoted normally to control renal phosphate reabsorption.

Oncogenic osteomalacia has been linked to secretion of a Frizzled receptor protein (FRP4) containing cysteine rich ligand binding domain as well’s hydrophilic C terminal region. The normally bound receptor link Wnt proteins in tandem with LPR family co-receptors. The binding to Wnt proteins to frizzled receptors and LPR5/6 coreceptors in heterotrimeric complexes on the cell surfaces leads to stabilization of intrcellular catenin beta and a complex network of singalling cascade.

It has been demonstrated in many cancers both in vivo and in vitro, that this pathways account for bone osteolysis in cancer diffusion to bone tissues.

Moreover some rare inherited disorders are characterized by involvement of Wnt/LPR pathways alterations:

. osteoporosis pseudoglioma syndrome : congenital blindness and severe chilhood osteoporosis

. High Bone Mass syndrome: associated with phenotypical presence of inherited high bone mass level.

FRP4 is located on chromosome 7p14.1 and it is conprised of six encoding exons, spanning 10.8 kb of genomic sequence. The translated protein product consist of 346 aminoacids, of which the first 21 residues constitute the predicted signal peptide. Finally the molecular mass of FRP4 is approximately of 40 kDa, but it is glycosylated to form a mature peptide of 48 kDa. It is ubiquitariously expressed, but importantly on bone cells it is present indicating a possible auto and paracrine effect in the skeleton too.

Concerning phosphate metabolism the study of Berndt et al (2003) revealed that this peptide has the capacity of inhibit the sodium dependent phosphate reuptake in opossum kidney in vitro experiments. Moreover infusion of FRP4 in vivo in parathyroidectomized mice caused an increase in the fractional excretion of phosphate and subsequent hypophosphatemia, indicating a mechansim of action partially independent from PTH.

Hyperphosphatemic Familial Tumoral Calcinosis

Recent work regarding fibroblast growth factor 23 addressed the question of pathogenesis of familial tumoral calcinosis. A missense mutation in the gene encoding FGF23 cause autosomal dominant hypophophatemic rickets. In addition FGF23 is highly expressed by tumors causing oncogenic hypophophatemic osteomalacia. FGF23 is therefore a strong candidate for “phophatonin”, the factor implicated as a cause of the phophate wasting in patients with oncogenic hypophosphatemic osteomalacia. The mutations tht cause autosomal dominant hypophosphatemic rickets stabilizes FGF23, potentially elevating its concentration in serum and leading to renal phosphate wasting. The same would be true in oncogenic hypophosphatemic osteomalacia: i.e. a great production of FGF23 by tumor and so increased levels of plasma FGF.

The clinical response to octreotide therapy as described by Seufert J in a patients may suggest that secretion of fibroblast growth factor 23 by the tumor can be modulated throught the somatostatin receptor signaling pathway. This protein (FGF23) has been demonstrated to be a substrate for the endopeptidase PHEX and to inhibit the phosphate transport in kidney cells. Moreover in autosomal dominant form of hypophosphatemic rickets, mutations in FGF23 have been identified that render the molecule resistant to cleavage by PHEX. So that in these patients octreotide scanning may be useful in identifying such a small tumors or relapses.

 

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NHERF

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