The retinoic acid receptors α, β and γ (RARα, RARβ and RARγ) are nuclear hormone receptors that regulate fundamental processes during embryogenesis, but their roles in skeletal development and growth remain unclear. To study skeletal-specific RAR function, we created conditional mouse mutants deficient in RAR expression in cartilage. We find that mice deficient in RARα and RARγ (or RARβ and RARγ) exhibit severe growth retardation obvious by about 3 weeks postnatally. Their growth plates are defective and, importantly, display a major drop in aggrecan expression and content. Mice deficient in RARα and RARβ, however, are virtually normal, suggesting that RARγ is essential. In good correlation, we find that RARγ is the most strongly expressed RAR in mouse growth plate and its expression characterizes the proliferative and pre-hypertrophic zones where aggrecan is strongly expressed also. By being avascular, those zones lack endogenous retinoids and thus RARγ is likely to exert ligand-less repressor function. Indeed, our data indicate that: aggrecan production is enhanced by RARγ over-expression in chondrocytes under retinoid-free culture conditions; production is further boosted by corepressor Zac1 or pharmacologic agents that enhance RAR repressor function; and RAR/Zac1 function on aggrecan expression may involve Sox proteins. In sum, our data reveal that RARs, and RARγ in particular, exert previously unappreciated roles in growth plate function and skeletal growth and regulate aggrecan expression and content. Since aggrecan is critical for growth plate function, its deficiency in RAR-mutant mice is likely to have contributed directly to their growth retardation.

SummaryWe investigated the molecular bases of spondyloepiphyseal dysplasia (SED) associated with the R992C (p.R1192C) substitution in collagen II. At the protein level we analyzed the structure and integrity of mutant molecules, and at the cellular level we specifically studied the effects of the presence of the R992C collagen II on the biological processes taking place in host cells. Our studies demonstrated that mutant collagen II molecules were characterized by altered electrophoretic mobility, relatively low thermostability, the presence of atypical disulfide bonds, and slow rates of secretion into the extracellular space. Analyses of cellular responses to the presence of the mutant molecules showed that excessive accumulation of thermolabile collagen II was associated with the activation of an "unfolded protein response" and an increase in apoptosis of host cells. Collectively, these data suggest that molecular mechanisms of SED may be driven not only by structural changes in the architecture of extracellular collagenous matrices, but also by intracellular processes activated by the presence of mutant collagen II molecules.

We investigated the effects of the presence of R75C (p.R275C), R519C (p.719C), R789C (p.R989C), and G853E (p.G1053E) type II collagen (COL2A1) mutants, associated with distinct forms of spondyloepiphyseal dysplasia (SED), on the biological processes occurring in chondrocytic cells harboring those mutants. Mutant-specific biological responses of cells were initiated by activating tetracycline (Tet)-dependent expression of type II collagen mutants. Employing microscopic and biochemical assays, we determined that cells expressing the thermolabile R789C (p.R989C) type II collagen mutant undergo apoptosis. In contrast, in cells expressing the thermostable R75C (p.R275C), R519C (p.719C), and G853E (p.G1053E) mutants, apoptotic markers were not apparent. We also demonstrated that the R789C (p.R989C) mutant formed atypical complexes with endoplasmic reticulum (ER)-resident chaperones, thereby indicating an "unfolded protein response" (UPR) of cells harboring this specific mutant. Apoptotic changes were also demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and cleaved caspase 3 assays in the growth plates of mice harboring the R992C (p.R1147C) substitution in type II collagen. Based on these results, we propose that the intracellular presence of structurally altered type II collagen mutants could activate an apoptotic response, thereby limiting cell survival. By analyzing the response of cells to the altered structure of collagen mutants, our study contributes to better understanding the molecular basis of the pathological changes seen in vivo at the tissue level.

Platelet glycoprotein VI (GPVI) is a key receptor for collagens that mediates the propagation of platelet attachment and activation. Targeted disruption of the murine gene Gp6 on a mixed 129 ؋ 1/ SvJ ؋ C57BL/6J background causes the expected defects in collagen-dependent platelet responses in vitro. The extent of this dysfunction in all Gp6 ؊/؊ mice is uniform and is not affected by genetic background. However, the same Gp6 ؊/؊ mice exhibit 2 diametrically opposed phenotypes in vivo. In some mice, tail bleeding times are extremely prolonged, and thrombus formation in an in vivo carotid artery ferric chloride-injury model is significantly impaired. In other littermates, tail bleeding times are within the range of wild-type mice, and in vivo thrombus formation is indistinguishable from that of control mice. Directed intercrosses revealed that these phenotypes are heritable, and a genome-wide singlenucleotide polymorphism scan revealed the most significant linkage to a single locus (8 megabases) on chromosome 4 (logarithm of the odds [LOD] score ‫؍‬ 6.9, P < .0001) that we designate Modifier of hemostasis (Mh) . IntroductionGlycoprotein VI (GPVI), which is restricted in expression to megakaryocytes and platelets, is an important receptor for collagens. GPVI (60-65 kDa) is noncovalently associated with the fragment crystallizable receptor ␥ (FcR␥) subunit, an immunoreceptor tyrosine-based activation motif (ITAM)-based signaling coreceptor, 1,2 and in the mouse, surface GPVI expression requires coexpression of FcR␥. 3 Upon engagement of collagens, the GPVI-FcR␥ complex transduces outside-in signals that involve spleen tyrosine kinase (Syk), linker for the activation of T cells (LAT), and Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76), resulting in phospholipase ␥2 and phosphatidylinositol 3-kinase activation, leading to release of granule contents, prothrombinase activity, and platelet aggregation. [4][5][6][7] Targeted gene deletion in the mouse is one strategy to generate experimental models to assess the role of membrane receptors in hemostasis and thrombosis. We were the first to report the targeted disruption of murine Gp6 8 and demonstrated, using functional in vitro assays, that platelets from homozygous Gp6 Ϫ/Ϫ mice fail to aggregate upon stimulation with type I fibrillar collagen and fail to form thrombi upon perfusion over collagen-coated surfaces. However, the in vivo tail bleeding times for the same Gp6 Ϫ/Ϫ mice were more often within the range seen with wild-type littermates (45-140 seconds), and only a minority of the Gp6 Ϫ/Ϫ F2 littermates (3/13) exhibited an abnormal, extremely prolonged tail bleeding time (Ͼ 600 seconds). Similar proportions of normal and abnormal mice have been observed in studies of genetically engineered FcR␥ Ϫ/Ϫ mice, in which platelet surface GPVI expression is ablated, or wild-type mice made GPVI-deficient after 7 days by intraperitoneal injection of rat anti-mouse GPVI monoclonal antibody JAQ1. [9][10][11] On repeated examination, we dete...

Mutations in COL2A1 produce a spectrum of disorders whose hallmark feature is alterations in skeletal development. Attempts to counteract the effects of collagen mutations at the molecular level have been relatively ineffective due to the inability to selectively suppress a mutant allele, and failure to deliver a sufficient number of cells expressing wild-type collagen. Moreover, these approaches are hampered because the minimal therapeutic conditions that would allow extracellular matrix remodeling and recovery of cells from stress are not known. Here, we employed a tetracycline-inducible system for expressing the R789C or R992C collagen II mutants, allowing us to decrease the production of mutant proteins by 25, 50, 75, or 100% with respect to their initial production. Through analysis of intracellular and extracellular parameters we have shown that affected cell/matrix systems are able to recover from mutation-induced aberrations only when 100% expression of mutant collagens is shut off, but not if the expression of small amounts of mutant molecules persists in the system. Our data suggest that efficient remodeling of tissues affected by the presence of thermolabile collagen mutants may depend on their complete elimination rather than on partial reduction.