Modulation of BMP-7 signaling pathway in chondrocytes by CD44 and hyaluronan

CD44 receptors on articular chondrocytes bind hyaluronan and maintain cartilage homeostasis by retaining and anchoring the proteoglycan aggregates in the pericellular matrix. Bone morphogenetic protein-7 (BMP-7) regulates cellular metabolism in embryonic and adult tissues, including cartilage development and homeostasis. BMP-7 generates an anabolic response in articular chondrocytes upregulating CD44, aggrecan, hyaluronan synthase-2 and type II collagen expression. Signal transduction occurs through phosphorylation and subsequent nuclear translocation of Smad1 and Smad4. A yeast-two hybrid screen revealed an interaction between Smad1 and CD44. Co-immunoprecipitation studies confirmed this interaction, which was reduced after BMP-7 stimulation. The disruption of hyaluronan binding to CD44, either by Streptomyces hyaluronidase treatment or over-expression of the C-terminal truncation mutants CD44HDelta67 or CD44HDelta54, resulted in diminished nuclear translocation of endogenous Smad1 in response to BMP-7. Other measures of the BMP-7 response by chondrocytes, namely Smad1 phosphorylation and activity of pGL3ti(SBE) 4-luciferase reporter construct, were decreased after pre-treatment with Streptomyces hyaluronidase; and Smad1 phosphorylation and nuclear translocation was restored upon addition of exogenous hyaluronan. However, the nuclear translocation of Smad2 in chondrocytes in response to TGF-beta1 was not diminished following disruption of the hyaluronan-dependent pericellular matrix. Disruption of actin cytoskeleton also inhibited nuclear translocation of Smad1 in response to BMP-7 but not of Smad2 in response to TGFbeta1. These data support a functional link between the BMP-7 signal transduction cascade and CD44, and also that hyaluronan-CD44 interactions promote the cellular response to BMP-7. Since BMP-7 is known to upregulate the mRNA levels of CD44, the potential BMP-7 responsive enhancer region in the CD44 gene was investigated. Of the several CD44 constructs generated, the pCD44(499)-Luc promoter construct was most responsive to BMP-7 and upon transfection in chondrocytes displayed a reduced BMP-7 response upon removal of CD44-hyaluronan binding. This suggests that hyaluronan-cell interactions are required for the BMP-7 mediated upregulation of CD44 gene expression. These results indicate that the regulation of functional CD44 expression and CD44 receptor ligation by hyaluronan influence physiological functions for CD44-Smadl interaction to modulate the cellular response to BMP-7. Thus modulation or disruption of hyaluronan-cell interactions during development, tissue remodeling or degeneration may alter cellular BMP responsiveness.