Cyclic mechanical strain regulates the PTHrP expression in cultured chondrocytes via activation of the Ca2+ channel.

The association between mechanical stimulation and chondrocyte homeostasis has been reported. However, the participation of PTHrP (parathyroid-hormone-related protein) in the mechano-regulation of chondrocyte metabolism remains unclear. We determined whether mechanical stimulation of chondrocytes induces the expression of PTHrP and, further, whether the mechano-modulation of PTHrP is dependent on the maturational status of chondrocytes. Cyclic mechanical strain was applied to rat growth plate chondrocytes at the proliferating, matrix-forming, and hypertrophic stages at 30 cycles/min. Cyclic mechanical strain significantly increased PTHrP mRNA levels in chondrocytes at the proliferating and matrix-forming stages only. The induction of PTHrP was dependent on loading magnitude at the proliferating stage. Using specific ion channel blockers, we determined that mechano-induction of PTHrP was inhibited by nifedipine, a Ca2+ channel blocker. These results suggest that mechanical induction of PTHrP possibly provides the environment for greater chondrocyte replication and matrix formation that would subsequently affect cartilage formation.

Three-dimensional structural analysis of fibronectin heparin-binding domain mutations.

Using recombinant fibronectin proteins containing the V region and two point mutations in the high-affinity heparin-binding domain, we previously showed that these domains modulate tumor cell invasion as well as proteinase expression and apoptosis in human fibroblasts. Structurally, the wildtype counterparts to these two point mutations, together with four other discontinuous, positively charged residues, form a cationic cradle in domain III-13 of fibronectin that binds heparin. We constructed a three-dimensional model of this cationic cradle and determined whether the two engineered point mutations in the heparin-binding domain would alter this cradle conformation, thus explaining the altered cell behavior. Our model of fibronectin domain III-13 was generated from a template of the three-dimensional structure of a homologous (25% identity) domain, III-3, from tenascin. The amino acid sequences of III-13 that differed from tenascin III-3 were replaced, and side chains for positively charged arginines 6 and 7 were substituted with uncharged threonines. The model revealed that the two mutated threonine residues were solvent accessible, readily accommodated as part of an antiparallel beta strand, and remained part of the three-dimensional cradle. These models suggest that the two point mutations in the heparin-binding domain of fibronectin III-13 alter cell function probably through changes in charge and not through changes in the conformational structure of the cationic cradle. J. Cell. Biochem. Suppl. 36: 156-161, 2001.