Murine dystrophia muscularis-2J (dy2J) is an autosomal recessive disorder characterized by muscular dystrophy and dysmyelination of peripheral nerve. Biochemical characterization of dy2J mice revealed the expression of a mutant laminin alpha 2 chain with a smaller molecular weight in the basal lamina of striated muscle and peripheral nerve. DNA sequencing of the alpha 2 chain cDNA amplified by RT-PCR from dy2J mice identified a novel and predominant transcript with a 171 base in-frame deletion. We also confirmed an underlying splice donor site mutation in the alpha 2 chain gene of the dy2J mouse. Translation of this variant transcript would result in the expression of a truncated alpha 2 chain having a 57 amino acid deletion (residues 34-90) and a substitution of Gln91Glu in the N-terminal domain VI, which is presumed to be involved in self-aggregation of laminin heterotrimers. Thus, the mutant alpha 2 chain could disrupt the formation of the laminin network and lead to muscle cell degeneration. Our results provide a molecular basis of muscular dystrophy and dysmyelination of peripheral nerve.

Elevated levels of CHI3L1 (chitinase-3-like protein 1) are associated with disorders exhibiting increased connective tissue turnover, such as rheumatoid arthritis, osteoarthritis, scleroderma, and cirrhosis of the liver. This secreted protein is not synthesized in young healthy cartilage, but is produced in cartilage from old donors or patients with osteoarthritis. The molecular processes governing the induction of CHI3L1 are currently unknown. To elucidate the molecular events involved in CHI3L1 synthesis, we investigated two models of articular chondrocytes: neonatal rat chondrocytes, which do not express CHI3L1, and human chondrocytes, which express CHI3L1 constitutively. In neonatal rat chondrocytes, the inflammatory cytokines tumor necrosis factor-␣ (TNF-␣) and interleukin-1 potently induced steady-state levels of CHI3L1 mRNA and protein secretion. Treatment of chondrocytes with TNF-␣ for as little as 1 h was sufficient for sustained induction up to 72 h afterward. Using inhibitors selective for the major signaling pathways implicated in mediating the effects of TNF-␣ and interleukin-1, only inhibition of NF-B activation was effective in curtailing cytokine-induced expression, including after removal of the cytokine, indicating that induction and continued production of CHI3L1 are controlled mainly by this transcription factor. Inhibition of NF-B signaling also abolished constitutive expression by human chondrocytes. Thus, induction and continued secretion of CHI3L1 in chondrocytes require sustained activation of NF-B. Selective induction of CHI3L1 by cytokines acting through NF-B coupled with the known restriction of the catabolic responses by CHI3L1 in response to these inflammatory cytokines represents a key regulatory feedback process in controlling connective tissue turnover.

After publication of our recent article [1], it has been brought to our attention that four panels in Figure 1 have been mislabeled. Images (c) and (e) are femurs, rather than tibias. Similarly, images (d) and (f) should be labelled as tibias.As such the figure legend should read as follows:

Tumor necrosis factor alpha (TNFalpha) inhibits matrix synthesis by chondrocytes in rheumatoid arthritis and osteoarthritis; however, the underlying signaling pathways are poorly characterized. This study investigated the TNFalpha-activated pathways regulating expression of two key components of the cartilage matrix-link protein and type II collagen. In rat articular chondrocytes, TNFalpha decreased link protein and type II collagen mRNA to undetectable levels within 48 h. Levels of link protein mRNA recovered more readily than type II collagen mRNA following removal of the cytokine. TNFalpha-mediated reduction in mRNA of both matrix molecules occurred at the level of transcription and, for link protein, mRNA stability. Turnover of type II collagen and link protein mRNA was dependent on new protein synthesis. In both prechondrocytes and articular chondrocytes, TNFalpha induced concentration-dependent activation of MEK1/2 and NF-kappaB, but not p38 or JNK. Sustained activation of NF-kappaB was observed for up to 72 h following continuous or transient exposure to TNFalpha. Using pharmacological and molecular approaches, the MEK1/2 and NF-kappaB pathways were found to mediate inhibition of type II collagen and link protein gene expression by TNFalpha. Both prechondrocytes and articular chondrocytes are targets of TNFalpha. This study identifies pathways through which TNFalpha perturbs the synthesis and organization of articular cartilage matrix during inflammation.

Objective. To define the roles of transforming growth factor ␣ (TGF␣) in cartilage degradation.Methods. Primary rat articular chondrocytes and articular osteochondral explants were cultured with TGF␣ to assess the effects of TGF␣ on chondrocyte physiology and phenotype.Results. TGF␣ altered chondrocyte morphology through reorganization of the actin cytoskeleton and formation of stress fibers. Expression of anabolic genes, including aggrecan, type II collagen, and cartilage link protein, was reduced in response to TGF␣. Proliferation of chondrocytes and formation of articular chondrocyte clusters was stimulated by TGF␣. Expression of matrix metalloproteinase 13 and cathepsin C was increased by TGF␣. We demonstrated the down-regulation of Sox9 messenger RNA and protein levels by TGF␣. This was associated with reduced levels of phosphorylated and total SOX9 in cartilage explants upon TGF␣ treatment. In contrast, another growth factor identified in our microarrays, Kitl, had no effects on the chondrocyte parameters tested. To examine correlations between the increased levels of TGF␣ in experimental knee osteoarthritis (OA) with the levels of TGF␣ in humans with knee OA, a microarray analysis of mRNA from 13 normal and 12 late-stage OA cartilage samples was performed. Seven OA samples showed TGFA mRNA levels similar to those in the normal controls, but expression was markedly increased in the other 5 OA samples. These data confirm that TGFA transcript levels are increased in a subset of patients with OA.Conclusion. This study adds TGF␣ to the list of dysregulated cytokines present in degrading cartilage in OA. Since TGF␣ inhibits articular chondrocyte anabolic capacity, increases catabolic factors, and contributes to the development of chondrocyte clusters, TGF␣ may be a potential target for therapeutic strategies in the treatment of OA.Articular cartilage degeneration is a defining feature of osteoarthritis (OA) (1). Under normal circumstances, chondrocytes are responsible for maintaining the cartilage extracellular matrix (ECM), which consists largely of type II collagen and proteoglycans. However, degenerative influences stimulate pathologic changes to the chondrocyte phenotype in OA. As a result, catabolic factors (e.g., matrix metalloproteinases [MMPs]) are synthesized and released from chondrocytes, weakening the ECM (2). These events make cartilage susceptible to swelling and mechanical disruption due to compromised structural integrity (3). OA chondrocytes also attempt to repair damaged tissues by chondrocyte proliferation (4), but repair is generally suboptimal.Cytokine release from articular chondrocytes is thought to promote cartilage degradation in OA. Numerous studies of interleukin-1␤ (IL-1␤) and tumor

Oculodentodigital dysplasia (ODDD) is associated with at least 28 connexin43 (Cx43) mutations. We characterized four of these mutants; Q49K, L90V, R202H, and V216L. Populations of these GFP-tagged mutants were transported to the cell surface in Cx43-negative HeLa cells and Cx43-positive NRK cells. Dual patch-clamp functional analysis in N2A cells demonstrated that channels formed by each mutant have dramatically reduced conductance. Dye-coupling analysis revealed that each mutant exhibits a dominant-negative effect on wild-type Cx43. Since ODDD patients display skeletal abnormalities, we examined the effect of three other Cx43 mutants previously shown to exert dominant-negative effects on wild-type Cx43 (G21R, G138R, and G60S) in neonatal calvarial osteoblasts. Differentiation was unaltered by expression of these mutants as alkaline phosphatase activity and extent of culture mineralization were unchanged. This suggests that loss-of-function Cx43 mutants are insufficient to deter committed osteoblasts from their normal function in vitro. Thus, we hypothesize that the bone phenotype of ODDD patients may result from disrupted gap junctional intercellular communication earlier in development or during bone remodeling.

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ABSTRACT:Introduction: Bone development and modeling requires precise gap junctional intercellular communication (GJIC). Oculodentodigital dysplasia (ODDD) is an autosomal dominant human disease caused by mutations in the gene (GJA1) encoding the gap junction protein, connexin43 (Cx43). The disease is characterized by craniofacial bone deformities and limb abnormalities. It is our hypothesis that Cx43 mutation causes osteoblast dysfunction, which may contribute to the bone phenotype of ODDD. Materials and Methods:We expressed human and mouse ODDD-linked Cx43 mutants in MC3T3-E1 cells and primary mouse osteoblasts by retroviral infection and evaluated their in vitro differentiation as an index of osteoblast function. We compared these findings to the differentiation of osteoblasts isolated from a mouse model of ODDD that harbors a germ line Cx43 mutation and exhibits craniofacial and limb defects mimicking human ODDD. We determined the differentiation status of osteoblasts by analyzing alkaline phosphatase activity and the expression levels of osteoblast markers including bone sialoprotein and osteocalcin. Results: We showed that ODDD-linked Cx43 mutants are loss-of-function and dominant-negative to coexpressed Cx43 and, furthermore, greatly inhibit functional GJIC in osteoblasts. Surprisingly, the mutants had only a minor effect on osteoblast differentiation when introduced into lineage committed cells. In contrast, osteoblasts isolated from the ODDD mouse model exhibited impaired late stage differentiation. Conclusions: Expression of human and mouse ODDD-linked Cx43 mutants failed to significantly impair differentiation in cells predisposed to the osteoblast lineage; however, germ line reduction of Cx43-based GJIC leads to impaired osteoblast differentiation, which may account for the bone phenotypes observed in ODDD patients.