Mutations in fibrillin-1 cause congenital scleroderma: stiff skin syndrome.

The predisposition for scleroderma, defined as fibrosis and hardening of the skin, is poorly understood. We report that stiff skin syndrome (SSS), an autosomal dominant congenital form of scleroderma, is caused by mutations in the sole Arg-Gly-Asp sequence-encoding domain of fibrillin-1 that mediates integrin binding. Ordered polymers of fibrillin-1 (termed microfibrils) initiate elastic fiber assembly and bind to and regulate the activation of the profibrotic cytokine transforming growth factor-beta (TGFbeta). Altered cell-matrix interactions in SSS accompany excessive microfibrillar deposition, impaired elastogenesis, and increased TGFbeta concentration and signaling in the dermis. The observation of similar findings in systemic sclerosis, a more common acquired form of scleroderma, suggests broad pathogenic relevance.

Extracellular microfibrils in development and disease.

Fibrillins are the structural components of extracellular microfibrils that impart physical properties to tissues, alone or together with elastin as elastic fibers. Genetic studies in mice have revealed that fibrillin-rich microfibrils are also involved in regulating developmental programs and homeostatic processes through the modulation of TGF-beta/BMP signaling events. A new paradigm has thus emerged whereby the spatiotemporal organization of microfibrils dictates both the cellular activities and physical properties of connective tissues. These observations have paved the way to novel therapeutic approaches aimed at counteracting the life-threatening complications in human conditions caused by dysfunctions of fibrillin-rich microfibrils.

Fibulin-5 deposition in human skin: decrease with ageing and ultraviolet B exposure and increase in solar elastosis.

BACKGROUND: Fibulin-5 was recently found as a secreted extracellular matrix protein that functions as a scaffold for elastic fibres. However, the distribution of fibulin-5 in human skin and its changes during the ageing process are not known. OBJECTIVES: To explore the involvement of fibulin-5 in skin ageing, the age-dependent changes in fibulin-5 localization in human skin were examined compared with those of other elastic fibre components including elastin, fibrillin-1 and fibulin-2. Methods The distribution of elastin, fibrillin-1, fibrillin-2, fibulin-2 and fibulin-5 was investigated by means of immunohistochemistry using their specific antibodies. Skin samples were recovered from 12 healthy subjects undergoing plastic surgery. Ultraviolet (UV) B-irradiated or control nonirradiated buttock skin samples were obtained from two healthy volunteers at 2 days after the irradiation at 2 minimal erythemal doses. RESULTS: In the reticular dermis of young sun-protected skin from the upper arm, fibulin-5 colocalized with the other elastic fibre components, while in the papillary dermis fibulin-5 showed candelabra-like structures perpendicular to the epidermis with an unstained area just beneath the epidermis, which was similar to that of elastin but not fibrillin-1. Fibulin-5 in the reticular dermis decreased and disappeared with age even in sun-protected skin from the thigh, abdomen and upper arm. In sun-exposed skin, fibulin-5 was extremely reduced in the dermis of cheek skin even from a 20-year-old man. UVB irradiation reduced fibulin-5, fibulin-2 and elastin markedly, moderately and weakly, respectively, compared with levels in control nontreated skin. Interestingly, the deposition of fibulin-5 was increased in solar elastosis, like that of other elastic fibre components. CONCLUSIONS: These results suggest that fibulin-5 is a good marker of skin ageing and that the earlier loss of fibulin-5 may involve age-dependent changes in other elastic fibre components.

Assembly of epithelial cell fibrillins.

Fibrillins are large structural macromolecules that are components of connective tissue microfibrils. Fibrillin microfibrils have been found in association with basement membranes, where microfibrils appear to insert directly into the lamina densa. It is unknown whether fibrillins are limited to these sites of microfibril insertion or are present throughout the lamina densa. In this study, electron microscopic immunolocalization demonstrated the presence of fibrillin-1 throughout the lamina densa in the dermal-- epidermal junction. In order to investigate whether fibrillin microfibrils might be present in the lamina densa, epithelial cell cultures (WISH, HaCaT, and primary keratinocytes) were analyzed by immunofluorescence, immunoblotting, and extraction of microfibrils followed by rotary shadowing electron microscopy and compared to mesenchymal cell cultures (dermal fibroblasts and MG63 osteosarcoma). In contrast to mesenchymal cells, which elaborate a fibrillin fibril network, epithelial cells primarily deposit fibrillin into the extracellular matrix in a nonfibrillar form. Coculture experiments using human epithelial cells and mouse fibroblasts implicated the cells themselves in the assembly of fibrillin. The importance of the cell in this process was further underscored by novel data demonstrating that keratinocytes selectively secrete fibrillin-1 into the matrix and not into the medium and can differentiate between fibrillin-1 and fibrillin-2.

Role of transforming growth factor-beta1 and its latent form binding protein in pseudoexfoliation syndrome.

Pseudoexfoliation (PEX) syndrome is a common and clinically important systemic condition characterized by the pathologic production and accumulation of an abnormal fibrillar extracellular material in many intra- and extraocular tissues. Recent evidence suggests that it is a type of elastosis associated with the excess synthesis of elastic microfibrillar components such as fibrillin-1. Since transforming growth factor (TGF)-beta is a major modulator of extracellular matrix formation, the potential involvement of TGF-beta and its latent form binding protein (LTBP) in this aberrant matrix process was investigated. The expression of various isoforms of TGF-beta and LTBP was investigated in the anterior segment tissues of PEX and control eyes on the protein and mRNA level by light and electron microscopic immunohistochemistry, in situ hybridization, and semiquantitative RT-PCR. TGF-beta1 and TGF-beta2 levels were measured in aqueous humor and serum of PEX and control patients by ELISA. Cultures of Tenon's capsule fibroblasts were established to study the effect of TGF-beta1 on fibrillin-1 mRNA expression by Northern blot analysis. Significantly increased concentrations of both total and active TGF-beta1 were measured in the aqueous humor of PEX eyes without and with glaucoma as compared to control eyes, whereas levels of TGF-beta2 were not significantly different. The expression of TGF-beta1, LTBP-1, and LTBP-2, but not TGF-beta2, was markedly increased in anterior segment tissues of PEX eyes, particularly in the non-pigmented epithelium of the ciliary body, on both the mRNA and the protein level. Latent TGF-beta1 staining was consistently associated with PEX material deposits and could be released by proteolytic processing. Double immunolabeling revealed clear co-localization of LTBP-1 and -2 with latent TGF-beta1 and with fibrillin-1 on PEX fibrils. The expression of mRNA coding for fibrillin-1 was up-regulated in vitro by TGF-beta1. This study provides evidence for a significant role of TGF-beta1 and the LTBPs 1 and 2 in PEX syndrome. The results suggest that increased levels of latent and active TGF-beta1 in the aqueous humor of PEX patients, derived from enhanced local synthesis and activation, promote the buildup of the abnormal extracellular elastic material characteristic of PEX syndrome. They further support a dual role for LTBPs, both as integral structural components of PEX fibers and as a means of matrix anchorage of latent TGF-beta1, representing one possible mechanism for the regulation of TGF-beta1 activity in PEX eyes. Future therapeutic strategies might focus on TGF-beta1 antagonistic approaches.

Fibrillin: from domain structure to supramolecular assembly.

In the last 5 years, significant progress has been made in understanding the structure and function of all the major domains composing the fibrillins. A previous review [Meth. Enzymol. 245 (1994), 29] focused on the isolation of fibrillin monomers and fibrillin-containing polymers (microfibrils). In this article, information gained from recent studies which have further elucidated molecular structure and investigated effects of mutations on structural and functional properties will be summarized. In addition, studies of functional domains in fibrillins which may be important in assembling microfibrils will be discussed. Throughout this review, the authors have attempted to identify areas of research which have been controversial. In the conclusion, we raise important questions which remain unresolved.

New insights into the assembly of extracellular microfibrils from the analysis of the fibrillin 1 mutation in the tight skin mouse.

The Tight skin (Tsk) mutation is a duplication of the mouse fibrillin 1 (Fbn1) gene that results in a larger (418 kD) than normal (350 kD) protein; Tsk/+ mice display increased connective tissue, bone overgrowth, and lung emphysema. Lung emphysema, bone overgrowth, and vascular complications are the distinctive traits of mice with reduced Fbn1 gene expression and of Marfan syndrome (MFS) patients with heterozygous fibrillin 1 mutations. Although Tsk/+ mice produce equal amounts of the 418- and 350-kD proteins, they exhibit a relatively mild phenotype without the vascular complications that are associated with MFS patients and fibrillin 1-deficient mice. We have used genetic crosses, cell culture assays and Tsk-specific antibodies to reconcile this discrepancy and gain new insights into microfibril assembly. Mice compound heterozygous for the Tsk mutation and hypomorphic Fbn1 alleles displayed both Tsk and MFS traits. Analyses of immunoreactive fibrillin 1 microfibrils using Tsk- and species-specific antibodies revealed that the mutant cell cultures elaborate a less abundant and morphologically different meshwork than control cells. Cocultures of Tsk/Tsk fibroblasts and human WISH cells that do not assemble fibrillin 1 microfibrils, demonstrated that Tsk fibrillin 1 copolymerizes with wild-type fibrillin 1. Additionally, copolymerization of Tsk fibrillin 1 with wild-type fibrillin 1 rescues the abnormal morphology of the Tsk/Tsk aggregates. Therefore, the studies suggest that bone and lung abnormalities of Tsk/+ mice are due to copolymerization of mutant and wild-type molecules into functionally deficient microfibrils. However, vascular complications are not present in these animals because the level of functional microfibrils does not drop below the critical threshold. Indirect in vitro evidence suggests that a potential mechanism for the dominant negative effects of incorporating Tsk fibrillin 1 into microfibrils is increased proteolytic susceptibility conferred by the duplicated Tsk region.

Mutations in calcium-binding epidermal growth factor modules render fibrillin-1 susceptible to proteolysis. A potential disease-causing mechanism in Marfan syndrome.

Most extracellular proteins consist of various modules with distinct functions. Mutations in one common type, the calcium-binding epidermal growth factor-like module (cbEGF), can lead to a variety of genetic disorders. Here, we describe as a model system structural and functional consequences of two typical mutations in cbEGF modules of fibrillin-1 (N548I, E1073K), resulting in the Marfan syndrome. Large (80-120 kDa) wild-type and mutated polypeptides were recombinantly expressed in mammalian cells. Both mutations did not alter synthesis and secretion of the polypeptides into the culture medium. Electron microscopy after rotary shadowing and comparison of circular dichroism spectra exhibited minor structural differences between the wild-type and mutated forms. The mutated polypeptides were significantly more susceptible to proteolytic degradation by a variety of proteases as compared with their wild-type counterparts. Most of the sensitive cleavage sites were mapped close to the mutations, indicating local structural changes within the mutated cbEGF modules. Other cleavage sites, however, were observed at distances beyond the domain containing the mutation, suggesting longer range structural effects within tandemly repeated cbEGF modules. We suggest that proteolytic degradation of mutated fibrillin-1 may play an important role in the pathogenesis of Marfan syndrome and related disorders.

Role of the latent transforming growth factor beta binding protein 1 in fibrillin-containing microfibrils in bone cells in vitro and in vivo.

Latent transforming growth factor beta-binding proteins (LTBPs) are extracellular matrix (ECM) proteins that bind latent transforming growth factor beta (TGF-beta) and influence its availability in bone and other connective tissues. LTBPs have homology with fibrillins and may have related functions as microfibrillar proteins. However, at present little is known about their structural arrangement in the ECM. By using antibodies against purified LTBP1, against a short peptide in LTBP1, and against epitope-tagged LTBP1 constructs, we have shown colocalization of LTBP1 and fibrillin 1 in microfibrillar structures in the ECM of cultured primary osteoblasts. Immunoelectron microscopy confirmed localization of LTBP1 to 10- to 12-nm microfibrils and suggested an ordered aggregation of LTBP1 into these structures. Early colocalization of LTBP1 with fibronectin suggested a role for fibronectin in the initial assembly of LTBP1 into the matrix; however, in more differentiated osteoblast cultures, LTBP1 and fibronectin 1 were found in distinct fibrillar networks. Overexpression of LTBP1 deletion constructs in osteoblast-like cells showed that N-terminal amino acids 67-467 were sufficient for incorporation into fibrillin-containing microfibrils and suggested that LTBP1 can be produced by cells distant from the site of fibril formation. In embryonic long bones in vivo, LTBP1 and fibrillin 1 colocalized at the surface of newly forming osteoid and bone. However, LTBP1-positive fibrils, which did not contain fibrillin 1, were present in cartilage matrix. These studies show that in addition to regulating TGF beta 1, LTBP1 may function as a structural component of connective tissue microfibrils. LTBP1 may therefore be a candidate gene for Marfan-related connective tissue disorders in which linkage to fibrillins has been excluded.

Initial steps in assembly of microfibrils. Formation of disulfide-cross-linked multimers containing fibrillin-1.

Fibrillins are the major constituents of extracellular microfibrils. How fibrillin molecules assemble into microfibrils is not known. Sequential extractions and pulse-chase labeling of organ cultures of embryonic chick aortae revealed rapid formation of disulfide-cross-linked aggregates containing fibrillin-1. These results demonstrated that intermolecular disulfide bond formation is an initial step in the assembly process. To identify free cysteine residues available for intermolecular cross-linking, small recombinant peptides of fibrillin-1 harboring candidate cysteine residues were analyzed. Results revealed that the first four cysteine residues in the unique N terminus form intramolecular disulfide bonds. One cysteine residue (Cys(204)) in the first hybrid domain of fibrillin-1 was found to occur as a free thiol and is therefore a good candidate for intermolecular disulfide bonding in initial steps of the assembly process. Furthermore, evidence indicated that the comparable cysteine residue in fibrillin-2 (Cys(233)) also occurs as a free thiol. These free cysteine residues in fibrillins are readily available for intermolecular disulfide bond formation, as determined by reaction with Ellman's reagent. In addition to these major results, the cleavage site of the fibrillin-1 signal peptide and the N-terminal sequence of monomeric authentic fibrillin-1 from conditioned fibroblast medium were determined.

Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1.

Dissecting aortic aneurysm is the hallmark of Marfan syndrome (MFS) and the result of mutations in fibrillin-1, the major constituent of elastin-associated extracellular microfibrils. It is yet to be established whether dysfunction of fibrillin-1 perturbs the ability of the elastic vessel wall to sustain hemodynamic stress by disrupting microfibrillar assembly, by impairing the homeostasis of established elastic fibers, or by a combination of both mechanisms. The pathogenic sequence responsible for the mechanical collapse of the elastic lamellae in the aortic wall is also unknown. Targeted mutation of the mouse fibrillin-1 gene has recently suggested that deficiency of fibrillin-1 reduces tissue homeostasis rather than elastic fiber formation. Here we describe another gene-targeting mutation, mgR, which shows that underexpression of fibrillin-1 similarly leads to MFS-like manifestations. Histopathological analysis of mgR/mgR specimens implicates medial calcification, the inflammatory-fibroproliferative response, and inflammation-mediated elastolysis in the natural history of dissecting aneurysm. More generally, the phenotypic severity associated with various combinations of normal and mutant fibrillin-1 alleles suggests a threshold phenomenon for the functional collapse of the vessel wall that is based on the level and the integrity of microfibrils.

Increased expression of fibrillin-1 in human corneas with bullous keratopathy.

PURPOSE: To characterize the expression of fibrillins, microfibril components, in human corneas with pseudophakic/aphakic (PBK/ABK) bullous keratopathy. METHODS: Normal and PBK/ABK corneas were stained by immunofluorescence for fibrillin-1 and -2. The expression of fibrillin-1 messenger RNA (mRNA) was studied by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Southern analysis. RESULTS: Only fibrillin-1 was detected in normal and diseased corneas. As described previously, in normal corneas, it was found in the limbal stroma and basement membrane (BM) and in the peripheral corneal epithelial BM for a short distance near the limbus. Central corneal BM, stroma, and Descemet's membrane were negative. All PBK/ABK corneas were positive for fibrillin-1, which was detected in fibrillar deposits at the endothelial face of Descemet's membrane, in the epithelial BM, subepithelial fibrosis areas, and posterior collagenous layer. By RT-PCR, low levels of fibrillin-1 mRNA were detected in normal corneas, and they increased significantly in PBK/ABK corneas. CONCLUSION: The deposition of fibrillin-1, together with tenascin-C, in PBK/ABK corneas may be part of an abnormal fibrotic/wound-healing process that occurs during the development of postsurgical corneal edema with the formation of bullae and posterior collagenous layer.

Targetting of the gene encoding fibrillin-1 recapitulates the vascular aspect of Marfan syndrome.

Aortic aneurysm and dissection account for about 2% of all deaths in industrialized countries; they are also components of several genetic diseases, including Marfan syndrome (MFS). The vascular phenotype of MFS results from mutations in fibrillin-1 (FBN1), the major constituent of extracellular microfibrils. Microfibrils, either associated with or devoid of elastin, give rise to a variety of extracellular networks in elastic and non-elastic tissues. It is believed that microfibrils regulate elastic fibre formation by guiding tropo-elastin deposition during embryogenesis and early post-natal life. Hence, vascular disease in MFS is thought to result when FBN1 mutations preclude elastic fibre maturation by disrupting microfibrillar assembly. Here we report a gene-targetting experiment in mice that indicates that fibrillin-1 microfibrils are predominantly engaged in tissue homeostasis rather than elastic matrix assembly. This finding, in turn, suggests that aortic dilation is due primarily to the failure by the microfibrillar array of the adventitia to sustain physiological haemodynamic stress, and that disruption of the elastic network of the media is a secondary event.

Fibrillin-1 in human cartilage: developmental expression and formation of special banded fibers.

The molecular basis for Marfan's syndrome (MS), a heritable disorder of connective tissue, is now known to reside in mutations in FBN1, the gene for fibrillin-1. Classic phenotypic manifestations of MS include several skeletal abnormalities associated primarily with overgrowth of long bones. As a first step towards understanding how mutations in FBN1 result in skeletal abnormalities, the developmental expression of fibrillin-1 (Fib-1) in human skeletal tissues is documented using immunohistochemistry and monoclonal antibodies demonstrated here to be specific for Fib-1. At around 10-11 weeks of fetal gestation, Fib-1 is limited in tissue distribution to the loose connective tissue surrounding skeletal muscle and tendon in developing limbs. By 16 weeks, Fib-1 is widely expressed in developing limbs and digits, especially in the perichondrium, but it is apparently absent within cartilage matrix. Fib-1 appears as a loose meshwork of fibers within cartilage matrix by 20 weeks of fetal gestation. Until early adolescence, Fib-1 forms loose bundles of microfibrils within cartilage. However, by late adolescence, broad banded fibers composed of Fib-1 are found accumulated pericellularly within cartilage. Because these fibers can be extracted from cartilage using dissociative conditions, we postulate that they are laterally packed and crosslinked microfibrils. On the basis of these findings, we suggest that the growth-regulating function of Fib-1 may reside persistently within the perichondrium. In addition, the accumulation of special laterally crosslinked Fib-1 microfibrils around chondrocytes during late adolescence suggests that growth-regulating activities may also be performed by Fib-1 at these sites.

Immunodissection of the connective tissue matrix in human skin.

Much of what has been learned of the components and structure of human skin over the past few years has been accomplished with the aid of antibody technology. Antibodies are used in techniques such as affinity chromatography to isolate individual molecules and by immunofluorescence and immunoelectron microscopy to identify each of those molecules as components of specific macromolecular assemblies present within the dermis. This manuscript is meant not as a review of technique but instead as a summary of recent progress made in the understanding of dermal matrix architecture.

Increased extracellular deposition of fibrillin-containing fibrils in pseudoexfoliation syndrome.

PURPOSE: To localize the distribution of fibrillin-containing microfibrils in normal human anterior segment tissues and to characterize the role of fibrillin in the pathogenesis of pseudoexfoliation syndrome. METHODS: Anterior segment tissues were obtained from 10 eyes with pseudoexfoliation syndrome and 10 normal eyes and investigated by indirect immunofluorescence and electron microscopic immunogold labeling using a monoclonal antibody to fibrillin-1. RESULTS: In addition to labeling of zonular fibers, fibrillin-immunoreactive microfibrillar bundles generally were found in the corneal stroma; the stromal connective tissues of conjunctiva, ciliary body, and iris, especially in the iris root area; the periphery of Schlemm's canal, the scleral spur, and the most anterior portion of the trabecular meshwork; the ciliary muscle, and the dilator and sphincter muscles of the iris; the basement membranes of peripheral corneal epithelium, conjunctival epithelium, ciliary pigmented epithelium, and the lens capsule. The microfibrillar bundles were found to be isolated or in association with elastic fibers and cellular basement membranes. In pseudoexfoliation eyes, an additional strong immunoreaction was localized to pseudoexfoliation fibers and their microfibrillar subunits in close proximity to surfaces of cells involved in pseudoexfoliation fiber production. CONCLUSIONS: The fibrillin-containing microfibrillar system in normal ocular tissues is suggested to have a substantial role in the maintenance of tissue integrity by providing tensile strength and flexibility to mechanically strained tissues. The findings further provide evidence for fibrillin as an intrinsic component of pseudoexfoliation fibers, suggesting the possibility that enhanced expression of fibrillin or abnormal aggregation of fibrillin-containing microfibrils may be involved in the pathogenesis of pseudoexfoliation syndrome.

Calcium determines the shape of fibrillin.

Velocity sedimentation experiments using authentic fibrillin-1 demonstrated sedimentation coefficients of s20,w0 = 5.1 +/- 0.1 in the Ca2+ form and s20,w0 = 6.2 +/- 0.1 in the Ca2+-free form. Calculations based on these results and the corresponding molecular mass predicted a shortening of fibrillin by approximately 25% and an increase in width of approximately 13-17% upon removal of Ca2+. These observations were confirmed by analysis of Ca2+-loaded and Ca2+-free rotary shadowed fibrillin molecules. Analysis of recombinant fibrillin-1 subdomain rF17, consisting primarily of an array of 12 Ca2+-binding epidermal growth factor (cbEGF)-like repeats, by analytical ultracentrifugation and rotary shadowing further confirmed Ca2+-dependent structural changes in the tertiary structure of fibrillin-1. Based on these results, the contribution of a single cbEGF-like repeat to the length of tandem arrays is predicted to be approximately 3 nm in the Ca2+ form. Ca2+-free forms demonstrated a decrease of 20-30% in length, indicating significant structural changes of these motifs when they occur in tandem. Circular dichroism measurements of rF17 in the presence and absence of Ca2+ indicated secondary structural changes within and adjacent to the interdomain regions that connect cbEGF-like repeats. The results presented here suggest a flexible structure for the Ca2+-free form of fibrillin which becomes stabilized, more extended, and rigid in the Ca2+ form.

Calcium stabilizes fibrillin-1 against proteolytic degradation.

The calcium-binding epidermal growth factor (cbEGF)-like domain is a structural motif that is present in many matrix proteins throughout the animal kingdom from invertebrates to mammals. This module has been demonstrated to bind calcium in the micromolar range. However, little is known about the functional consequences of calcium binding to proteins that contain this structural element. We used fibrillin-1, an extracellular matrix protein consisting of approximately 60% cbEGF-like motifs, as a model system to study stabilizing effects of calcium in protease degradation assays. Authentic human fibrillin-1 and recombinant human fibrillin-1 subdomains, spanning the whole molecule, showed significantly slower proteolytic degradation in the presence of CaCl2 than in the presence of EDTA, demonstrating that calcium stabilizes the structure of fibrillin-1 and protects the molecule against proteolytic degradation. Information about cleavage sites protected by calcium was obtained with a new recombinant subdomain, rF17 (Asp 952-Val 1527), comprising the longest stretch of cbEGF-like motifs in the center of the fibrillin-1 molecule. The most sensitive sites for trypsin and endoproteinase Glu-C were observed in cbEGF-like motifs 11 (Met 1034 and Asn 1046), 12 (Ser 1103), and 17 (Thr 1318). Since most of the currently known mutations in fibrillin-1 are found within cbEGF-like motifs and are predicted to disrupt calcium binding, we suggest that these mutations render fibrillin-1 more susceptible to proteolytic cleavage, and this might be one of the reasons why these mutations result in Marfan's syndrome.

The fate of cartilage oligomeric matrix protein is determined by the cell type in the case of a novel mutation in pseudoachondroplasia.

We have identified a novel missense mutation in a pseudoachondroplasia (PSACH) patient in one of the type III repeats of cartilage oligomeric matrix protein (COMP). Enlarged lamellar rough endoplasmic reticulum vesicles were shown to contain accumulated COMP along with type IX collagen, a cartilage-specific component. COMP was secreted and assembled normally into the extracellular matrix of tendon, demonstrating that the accumulation of COMP in chondrocytes was a cell-specific phenomenon. We believe that the intracellular storage of COMP causes a nonspecific aggregation of cartilage-specific molecules and results in a cartilage matrix deficient in required structural components leading to impaired cartilage growth and maintenance. These data support a common pathogenetic mechanism behind two clinically related chondrodysplasias, PSACH and multiple epiphyseal dysplasia.