|Budget Amount *help
¥3,100,000 (Direct Cost : ¥3,100,000)
Fiscal Year 2003 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 2002 : ¥2,400,000 (Direct Cost : ¥2,400,000)
This study aimed to clarify the molecular mechanisms of mechanosensitive responses in human umbilical vein endothelial cells (HUVECs). The results obtained in the present study are as follows ;
(1)Identification of Dbl protein involved in hypotonic stress-induced responses : It is known that the activation of Rho requires Dbl family protein. RT-PCR analysis revealed that HUVECs express mRNAs of S Dbl family proteins including Abr, Fgd-1, Kalirin-7, and Lbc. We examined the effects of the antisense oligonucleotides against these m-RNAs on hypotonic stress (HTS)-induced Ca^<2+> transients, and found that the antisense against Lbc suppressed it. Furthermore, overexpression of Lbc cDNA augments HTS-induced Ca^<2+> transients. These suggest that mechanical stress induces the activation of Rho in HUVECs via Lbc.
(2)Interrelation between HTS-induced tyrosine kinase activation and Rho activation : HTS-induced activation of two tyrosine kinases, FAK and paxillin, was detect with Western blotting. Tyrosine kinase inhibitors suppressed ATP release induced by lysophosphatidic acid (LPA), which activates Rho. Furthermore, HTS-induced tyrosine phosphorylation of FAK and paxillin was suppressed by Rho-kinase inhibitor Y27632. These indicate that HTS-induced responses are obtained by the sequential activation of FAK/paxillin followed by Rho/Rho-kinase.