Activation of K-p21ras and N-p21ras, but not H-p21ras, is necessary for mitogen-induced human airway smooth-muscle proliferation

Ras proteins (H-, K-, and N-p21ras) play critical roles in the control of normal and neoplastic cell growth. To date, however, little is known about the role of p21ras in regulating mitogen-induced smooth muscle and, specifically, human airway smooth-muscle (HASM) cell growth. We postulate that p21ras is a critical signaling event regulating mitogen-induced HASM cell proliferation. Growth-arrested, confluent HASM cells were treated for 1 h with 10 ng/ml epidermal growth factor (EGF), 1 U/ml thrombin, or 5 microM bradykinin, then cell lysates were immunoprecipitated using anti-p21ras antibody. Immunoblot analysis using a pan p21ras antibody, which recognizes H-, K-, and N-p21ras, found no significant difference in p21ras expression in HASM after stimulation with either agent, as compared with control. In parallel experiments, we characterized that HASM cells express K- and N-p21ras, but not H-p21ras. Further, there was no difference between the levels of each p21ras isoform after stimulation with any of the agonists. The time course of p21ras activation, however, was markedly different among agonists. EGF rapidly activated p21ras within 30 s and was sustained for up to 30 min. Although thrombin also induced a rapid rise in p21ras activity after 2.5 min, the activation was transient. In contrast, bradykinin, which is nonmitogenic for HASM cells, did not activate p21ras. Using single-cell microinjection, the role of p21ras activation in modulating mitogen-induced HASM DNA synthesis was determined by 5-bromo-2'-deoxyuridine (BrdU) incorporation and anti-BrdU immunofluorescent staining. Thrombin- and EGF-induced DNA synthesis in cells microinjected with Y13-259, a neutralizing p21ras antibody, was significantly inhibited as compared with those microinjected with isotype-matched rat immunoglobulin G(1) or a vehicle control. These data suggest that activation of p21ras appears to be necessary for EGF and thrombin-induced HASM cell proliferation and that activation of K- and N-p21ras, but not H-p21ras, mediates smooth-muscle cell growth.