Matrix metalloproteinase-13 influences ERK signalling in articular rabbit chondrocytes.

OBJECTIVE: Matrix metalloproteinase-13 (MMP-13) is an extracellular MMP that cleaves type II collagen, the major protein component of cartilage, with high specificity and has been implicated in the pathology of osteoarthritis. The present study aimed to characterize the binding and internalization kinetics of MMP-13 in normal rabbit chondrocytes and whether MMP-13 affected cell signalling. METHODS: Rabbit chondrocytes were used in [125I]-MMP-13 binding assays to investigate the MMP-13 binding kinetics and Western analysis allowed for the assessment of intracellular signalling cascades. RESULTS: Rabbit chondrocytes were found to express the cartilage-specific genes aggrecan and type II collagen throughout their in vitro culture period. Appreciable specific cell-association of [125I]-MMP-13 was detected after 10 min of exposure to the ligand and equilibrium was obtained after 2 h. Binding of [125I]-MMP-13 to chondrocytes was specific and approached saturation at 75 nM. Internalization of MMP-13 was evident after 20 min, reached a maximum at 30 min and had returned to baseline by 90 min. Addition of receptor-associated protein (RAP) inhibited the internalization of MMP-13 indicating a likely role for low-density lipoprotein receptor-related protein-1 (LRP1) in this process. Interestingly the presence of MMP-13 induced phosphorylation of the extracellular signal-regulated kinase 1/2 (ERK1/2) protein showing that there is initiation of a signalling process in response to MMP-13 being bound and internalized by rabbit chondrocytes. However, this activation does not involve the MMP-13 internalization receptor LRP1. CONCLUSION: These studies demonstrate and characterize the MMP-13 binding and internalization system in rabbit chondrocytes and indicate that MMP-13 may regulate the phenotype of the chondrocytes through this receptor system.

Isolation and characterization of a novel coactivator protein, NCoA-62, involved in vitamin D-mediated transcription.

The vitamin D receptor (VDR) forms a heterodimeric complex with retinoid X receptor (RXR) and binds to vitamin D-responsive promoter elements to regulate the transcription of specific genes or gene networks. The precise mechanism of transcriptional regulation by the VDR.RXR heterodimer is not well understood, but it may involve interactions of VDR.RXR with transcriptional coactivator or corepressor proteins. Here, a yeast two-hybrid strategy was used to isolate proteins that selectively interacted with VDR and other nuclear receptors. One cDNA clone designated NCoA-62, encoded a 62, 000-Da protein that is highly related to BX42, a Drosophila melanogaster nuclear protein involved in ecdysone-stimulated gene expression. Yeast two-hybrid studies and in vitro protein-protein interaction assays using glutathione S-transferase fusion proteins demonstrated that NCoA-62 formed a direct protein-protein contact with the ligand binding domain of VDR. Coexpression of NCoA-62 in a vitamin D-responsive transient gene expression system augmented 1, 25-dihydroxyvitamin D3-activated transcription, but it had little or no effect on basal transcription or gal4-VP16-activated transcription. NCoA-62 also interacted with retinoid receptors, and its expression enhanced retinoic acid-, estrogen-, and glucocorticoid-mediated gene expression. These data indicate that NCoA-62 may be classified into an emerging set of transcriptional coactivator proteins that function to facilitate vitamin D- and other nuclear receptor-mediated transcriptional pathways.

The N-terminal domain of transcription factor IIB is required for direct interaction with the vitamin D receptor and participates in vitamin D-mediated transcription.

The interaction of the vitamin D receptor (VDR) with transcription factor IIB (TFIIB) represents a potential physical link between the VDR-DNA complex and the transcription preinitiation complex. However, the functional relevance of the VDR-TFIIB interaction in vitamin D-mediated transcription is not well understood. In the present study, we used site-directed mutagenesis to demonstrate that the structural integrity of the amino-terminal zinc finger of TFIIB is essential for VDR-TFIIB complex formation. Altering the putative zinc-coordinating residues (C15, C34, C37, or H18) to serines abolished TFIIB interaction with the VDR as assessed in a yeast two-hybrid system and by in vitro protein interaction assays. This N-terminal, VDR-interactive domain functioned as a selective, dominant-negative inhibitor of vitamin D-mediated transcription. Expressing amino acids 1-124 of human TFIIB (N-TFIIB) in COS-7 cells or in osteoblastic ROS17/2.8 cells effectively suppressed 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-induced transcription, but had no effect on basal or glucocorticoid-activated transcription. A mutant N-terminal domain [N-TFIIB(C34S:C37S)] that does not interact with VDR had no impact on 1,25-(OH)2D3-induced transcription. Interestingly, both in vitro and in vivo protein interaction assays showed that the VDR-TFIIB protein complex was disrupted by the 1,25-(OH)2D3 ligand. Mechanistically, these data establish a functional role for the N terminus of TFIIB in VDR-mediated transcription, and they allude to a role for unliganded VDR in targeting TFIIB to the promoter regions of vitamin D-responsive target genes.

Ras-stimulated extracellular signal-related kinase 1 and RhoA activities coordinate platelet-derived growth factor-induced G1 progression through the independent regulation of cyclin D1 and p27.

Platelet-derived growth factor (PDGF)-induced Ras activation is required for G1 progression in Chinese hamster embryo fibroblasts (IIC9 cells). Ras stimulates both extracellular signal-related kinase (ERK) activation and RhoA activation in response to PDGF stimulation. Inhibition of either of these Ras-stimulated pathways results in growth arrest. We have shown previously that Ras-stimulated ERK activation is essential for the induction and continued G1 expression of cyclin D1. In this study we examine the role of Ras-induced RhoA activity in G1 progression. Unstimulated IIC9 cells expressed high levels of the G1 cyclin-dependent kinase inhibitor p27(KIP1). Stimulation with PDGF resulted in a dramatic decrease in p27(KIP1) protein expression. This decrease was attributed to increased p27(KIP1) protein degradation. Overexpression of dominant-negative forms of Ras or RhoA completely blocked PDGF-induced p27(KIP1) degradation, but only dominant-negative Ras inhibited cyclin D1 protein expression. C3 transferase also inhibited PDGF-induced p27(KIP1) degradation, thus further implicating RhoA in p27(KIP1) regulation. Overexpression of dominant-negative ERK resulted in inhibition of PDGF-induced cyclin D1 expression but had no effect on PDGF-induced p27(KIP1) degradation. These data suggest that Ras coordinates the independent regulation of cyclin D1 and p27(KIP1) expression by the respective activation of ERK and RhoA and that these pathways converge to determine the activation state of complexes of cyclin D1 and cyclin-dependent kinase in response to mitogen.

The vitamin D receptor interacts with general transcription factor IIB.

The vitamin D receptor (VDR) heterodimerizes with retinoid X receptors (RXR) on many vitamin D-responsive promoter elements, suggesting that this complex is the active factor in vitamin D-mediated transcription. However, the mechanism of transcriptional regulation following VDR-RXR binding to DNA is not well characterized. Using a yeast two-hybrid protein interaction assay, we demonstrate that VDR forms specific protein: protein contacts with the basal transcription factor TFIIB. Deletion analysis indicated that the carboxyl-terminal ligand binding domain of VDR interacted with a 43-residue amino-terminal domain in TFIIB. The interaction with TFIIB showed selectivity for the ligand binding domain of VDR as similar regions of RXR alpha or of retinoic acid receptor alpha did not couple with TFIIB. Binding assays with purified proteins showed a direct interaction between VDR and TFIIB in vitro. These data suggest a mechanism for VDR-dependent transcription in which protein contacts between VDR and TFIIB may impart regulatory information to the transcription preinitiation complex.