Arthur J. Cohen

The Transforming Growth Factor-β-inducible Matrix Protein βig-h3 Interacts with Fibronectin

Proper growth and development require the orderly synthesis and deposition of individual components of the extracellular matrix (ECM) into well ordered networks. Once formed, the ECM maintains tissue structure and houses resident cells. One ECM component, βig-h3, is a highly conserved transforming growth factor-β-inducible protein that has been hypothesized to function as a bifunctional linker between individual matrix components and resident cells. To gain insights into its physiological function, full-length βig-h3 protein was produced using a baculovirus expression system and purified under native conditions. Human fibroblasts attached and spread on βig-h3-coated plates and developed actin stress fibers. Purified βig-h3 binds fibronectin (FN) and type I collagen (Col I) but does not bind gelatin. Using defined fragments of FN, we localized the βig-h3 recognition region to the gelatin/collagen binding domain present in the N-terminal region of the FN molecule. Our results identify FN and Col I as two ligands of βig-h3 in the ECM.

Integration of Signaling Pathways Regulating Chondrocyte Differentiation During Endochondral Bone Formation

During endochondral bone formation, chondrocytes undergo a process of terminal differentiation or maturation, during which the rate of proliferation decreases, cells become hypertrophic, and the extracellular matrix is altered by production of a unique protein, collagen X, as well as proteins that promote mineralization. The matrix surrounding the hypertrophic chondrocytes eventually becomes mineralized, and the mineralized matrix serves as a template for bone deposition. This process is responsible for most longitudinal bone growth, both during embryonic development and in the postnatal long bone growth plates. Chondrocyte maturation must be precisely controlled, balancing proliferation with terminal differentiation; changes in the rate of either proliferation or differentiation result in shortened bones. Numerous signaling molecules have been implicated in regulation of this process. These include the negative regulators Indian hedgehog (Ihh) and parathyroid hormone‐related protein (PTHrP; Pthlh, PTH‐like hormone), as well as a number of positive regulators. This review will focus on several positive regulators which exert profound effects on chondrocyte maturation: the thyroid hormones T3 and T4, retinoic acid (the major active metabolite of vitamin A) and bone morphogenetic proteins (BMPs), as well as the transcription factor Runx2. Each of these molecules is essential for endochondral bone formation and cannot compensate for the others; abrogation of any one of them prevents differentiation. The important features of each of these signaling pathways will be discussed as they relate to chondrocyte maturation, and a model will be proposed suggesting how these pathways may converge to regulate this process. J. Cell. Physiol. 213:635–641.

The TGFβ-inducible matrix protein βig-h3 interacts with fibronectin

Proper growth and development require the orderly synthesis and deposition of individual components of the extracellular matrix (ECM) into well ordered networks. Once formed, the ECM maintains tissue structure and houses resident cells. One ECM component, βig-h3, is a highly conserved transforming growth factor-β-inducible protein that has been hypothesized to function as a bifunctional linker between individual matrix components and resident cells. To gain insights into its physiological function, full-length βig-h3 protein was produced using a baculovirus expression system and purified under native conditions. Human fibroblasts attached and spread on βig-h3-coated plates and developed actin stress fibers. Purified βig-h3 binds fibronectin (FN) and type I collagen (Col I) but does not bind gelatin. Using defined fragments of FN, we localized the βig-h3 recognition region to the gelatin/collagen binding domain present in the N-terminal region of the FN molecule. Our results identify FN and Col I as two ligands of βig-h3 in the ECM.

Thyroid hormone treatment of cultured chondrocytes mimics in vivo stimulation of collagen X mRNA by increasing BMP 4 expression

During endochondral bone formation, chondrocytes undergo terminal differentiation, during which the rate of proliferation decreases, cells become hypertrophic, and the extracellular matrix is altered by production of collagen X, as well as proteins required for matrix mineralization. This maturation process is responsible for most longitudinal bone growth, both during embryonic development and in postnatal long bone growth plates. Among the major signaling molecules implicated in regulation of this process are the positive regulators thyroid hormone (T3) and bone morphogenetic proteins (BMPs). Both T3 and BMPs are essential for endochondral bone formation and cannot compensate for each other, suggesting interaction of the two signaling pathways. We have analyzed the temporal and spatial expression patterns of numerous genes believed to play a role in chondrocyte maturation. Our results show that T3 stimulates collagen X gene expression in cultured chondrocytres with kinetics and magnitude similar to those observed in vivo. Stimulation of collagen X gene expression by T3 occurs only after a significant delay, implying that this hormone may act indirectly. We show further that T3 rapidly stimulates production of BMP 4, concomitant with a decrease in the BMP inhibitor Noggin, potentially resulting in a net increase in BMP signaling. Finally, inhibition of BMP signaling with exogenous Noggin prevents T3 stimulation of collagen X expression, indicating that BMP signaling is essential for this process. These data position thyroid hormone at the top of a T3/BMP cascade, potentially explaining why both pathways are essential for chondrocyte maturation. J. Cell. Physiol. 219: 595–605, 2009.

Retinoids directly activate the collagen X promoter in prehypertrophic chondrocytes through a distal retinoic acid response element

Retinoids are essential for the terminal differentiation of chondrocytes during endochondral bone formation. This maturation process is characterized by increased cell size, expression of a unique extracellular matrix protein, collagen X, and eventually by mineralization of the matrix. Retinoids stimulate chondrocyte maturation in cultured cells and experimental animals, as well as in clinical studies of synthetic retinoids; furthermore, retinoid antagonists prevent chondrocyte maturation in vivo. However, the mechanisms by which retinoids regulate this process are poorly understood. We and others showed previously that retinoic acid (RA) stimulates expression of genes encoding bone morphogenetic proteins (BMPs), suggesting that retinoid effects on chondrocyte maturation may be indirect. However, we now show that RA also directly stimulates transcription of the collagen X gene promoter. We have identified three RA response element (RARE) half‐sites in the promoter, located 2,600 nucleotides upstream from the transcription start site. These three half‐sites function as two overlapping RAREs that share the middle half‐site. Ablation of the middle half‐site destroys both elements, abolishing RA receptor (RAR) binding and drastically decreasing RA stimulation of transcription. Ablation of each of the other two half‐sites destroys only one RARE, resulting in an intermediate level of RAR binding and transcriptional stimulation. These results, together with our previously published data, indicate that retinoids stimulate collagen X transcription both directly, through activation of RARs, and indirectly, through increased BMP production. J. Cell. Biochem.

The Chick α2(I) Collagen Gene Contains Two Functional Promoters, and Its Expression in Chondrocytes Is Regulated at Both Transcriptional and Post-transcriptional Levels

Embryonic chick cartilages contain transcripts derived from the α2(I) collagen gene, although type I collagen is not normally found in these tissues; most of these RNAs are alternative transcripts initiating within intron 2. Use of the internal start site results in replacement of exons 1 and 2 with a previously undescribed exon and a change in the translational reading frame; thus, the alternative transcript cannot encode α2(I) collagen. We have demonstrated that production of the alternative transcript is due to activation of an internal promoter in chondrocytes and have identified a 179-base pair domain that is required for its activity. Furthermore, we have shown that the alternative transcript resulting from activation of the internal promoter turns over relatively rapidly; thus, the steady-state level of this transcript is less than predicted based on the transcription rate. The upstream promoter is only partially repressed in chondrocytes, suggesting that the lack of authentic α2(I) collagen mRNA may also be due in part to decreased mRNA stability. Thus, repression of α2(I) collagen synthesis in cartilage involves both transcriptional and post-transcriptional mechanisms. In contrast, repression of α1(I) collagen synthesis appears to be mediated primarily at the level of transcription.

Stimulation of Type-X Collagen Gene Transcription by Retinoids Occurs in Part Through the BMP Signaling Pathway

Background: Chondrocyte maturation and hypertrophy during endochondral bone formation are stimulated by both retinoids and bone morphogenetic proteins (BMPs). The type-X collagen gene, which is expressed only in hypertrophic chondrocytes, provides an excellent marker for chondrocyte maturation. We previously identified a 651-base-pair region of the type-X collagen promoter that is essential for its activation by BMP. We examined the relationship between the retinoid and BMP signaling pathways in transcriptional stimulation of the type-X collagen gene to determine whether they act independently or interact to regulate endochondral bone formation.Methods: Prehypertrophic chondrocytes from embryonic chick sterna cultured in the presence or absence of retinoic acid or BMP-2 were transiently transfected with plasmids containing various mutations of the type-X collagen promoter directing expression of a luciferase reporter gene. In addition, real-time polymerase chain reaction was used to examine the effects of retinoic acid on expression of genes encoding BMP-2, 4, and 6.Results: The previously identified BMP-responsive region of the type-X collagen promoter also mediated stimulation by physiological concentrations of retinoic acid in prehypertrophic chondrocytes. Systematic deletion mutagenesis of the BMP/retinoid-responsive region of the type-X collagen promoter identified distinct regions that are responsible for promoter stimulation by retinoids and BMP. Retinoic acid rapidly and dramatically stimulated accumulation of BMP-2 and BMP-6 messenger RNAs.Conclusions: These results suggest that, while retinoic acid appears to stimulate type-X collagen gene transcription in part by stimulating the BMP signaling pathway, it also acts in part through mechanisms that are independent of BMP.Clinical Relevance: Retinoids are essential for chondrocyte maturation during endochondral bone formation, but at high concentrations vitamin A is a potent teratogen; thus, both excessive and deficient vitamin A cause skeletal abnormalities by mechanisms that are not well understood. Since synthetic derivatives of vitamin A are widely used therapeutic agents for the treatment of several types of diseases, it is important to understand their effects on endochondral bone formation.

A novel noncollagenous protein encoded by an alternative transcript of the chick type III collagen gene is expressed in cartilage, bone and muscle.

We have identified a noncollagenous protein, Col3alt, encoded by an alternative transcript of the chick type III collagen gene; its amino acid sequence is out of frame with the collagen coding sequence. This 178-amino-acid protein is unique and has no recognizable motifs other than a hydrophobic domain. Col3alt is found in embryonic cartilage, muscle and bone and in the proliferative and prehypertrophic zones of juvenile chicken growth plates. The protein is intracellular in immature chondrocytes and myoblasts, but is extracellular in well-differentiated cartilage, muscle and bone, despite the lack of a conventional signal peptide. These results demonstrate an unexpected economy of genome utilization in which a single gene, using alternative promoters, gives rise to two unrelated proteins, type III collagen and Col3alt.