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Involvement of Rho p21 small GTP-binding protein and its regulator in the HGF-induced cell motility
1Department of Biochemistry, Kobe University School of Medicine, Japan.
Abstract:
Hepatocyte growth factor (HGF) induced motility of cultured mouse keratinocytes (308R cells). This HGF-induced cell motility was inhibited by microinjection of either rho GDI, an inhibitory GDP/GTP exchange protein for rho p21 small GTP-binding protein, or a botulinum exoenzyme C3 which is known to selectively impair the function of rho p21 by ADP-ribosylating its effector domain. The rho GDI action was prevented by comicroinjection with the guanosine 5'-(3-0-thio)triphosphate (GTP gamma S)-bound active form of rhoA p21, and the C3 action was prevented by comicroinjection with a rhoA p21 mutant (rhoAIle41 p21) which is resistant to the C3 action. The HGF-induced cell motility was not inhibited by microinjection of a dominant negative rac1 p21 mutant (rac1Asn17 p21) or a dominant negative Ki-ras p21 mutant (Ki-rasAsn17 p21). Microinjection of the GTP gamma S-bound form of rac1 p21 or a dominant active Ki-ras p21 mutant (Ki-rasVal12 p21) did not induce cell motility. These results indicate that both rho p21 and rho GDI, but neither rac p21 nor ras p21, are involved in the HGF-induced cell motility. However, microinjection of the GTP gamma S-bound form of rhoA p21 alone did not induce cell motility in the absence of HGF, suggesting that activation of rho p21 is necessary but not sufficient for the HGF-induced cell motility. The HGF-induced cell motility was mimicked by 12-0-tetradecanoyl-phorbol-13-acetate, a protein kinase C-activating phorbol ester, but not by Ca2+ ionophore. The phorbol ester-induced cell motility was also inhibited by microinjection of rho GDI or C3. These results indicate that both rho p21 and rho GDI are also involved in the phorbol ester-induced cell motility.
Insights
Hepatocyte growth factor (HGF) drives cell movement through the rho p21 pathway, not rac or ras. Rho p21 activation is essential but not sufficient for HGF-induced keratinocyte motility.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Hepatocyte growth factor (HGF) is a key regulator of cell growth, survival, and motility.
- Small GTP-binding proteins, including rho, rac, and ras, play critical roles in cellular signaling pathways that control cell behavior.
- Understanding the specific molecular mechanisms underlying HGF-induced cell motility is crucial for various biological processes, including development and wound healing.
Purpose of the Study:
- To elucidate the specific small GTP-binding proteins involved in Hepatocyte growth factor (HGF)-induced keratinocyte motility.
- To investigate the role of rho p21, rac p21, and ras p21 in HGF-mediated cell migration.
- To determine whether rho GDI and botulinum exoenzyme C3 can inhibit HGF-induced cell motility.
Main Methods:
- Microinjection of cultured mouse keratinocytes (308R cells) with various reagents, including rho GDI, botulinum exoenzyme C3, and specific mutants or active forms of rhoA, rac1, and Ki-ras p21.
- Assessment of HGF-induced cell motility.
- Investigation of the effects of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and Ca2+ ionophore on cell motility and the involvement of rho p21.
Main Results:
- HGF-induced keratinocyte motility was significantly inhibited by microinjection of rho GDI or botulinum exoenzyme C3, both of which target rho p21.
- The inhibitory effects of rho GDI and C3 were prevented by co-microinjection with active rhoA p21 or C3-resistant rhoA p21 mutants, respectively.
- Microinjection of dominant-negative rac1 or Ki-ras mutants, or active forms of rac1 or Ki-ras, did not affect HGF-induced motility, indicating their lack of involvement.
- While activation of rho p21 was necessary, it was not sufficient to induce motility in the absence of HGF.
- Phorbol ester (TPA)-induced cell motility, but not Ca2+ ionophore-induced motility, was also inhibited by rho GDI and C3, further implicating rho p21.
Conclusions:
- Rho p21 and its regulator rho GDI are critically involved in Hepatocyte growth factor (HGF)-induced keratinocyte motility.
- Neither rac p21 nor ras p21 play a significant role in HGF-induced cell migration.
- The signaling pathway involving rho p21 is essential for HGF-mediated cell motility, and its activation is a necessary but not sufficient event.
- The findings suggest that HGF activates rho p21 through a pathway that is also engaged by protein kinase C activators like TPA.