Hypoxia has been shown to act as a proliferative stimulus for adventitial fibroblasts of the pulmonary artery. The signaling pathways involved in this growth response, however, remain unclear. We tested the hypothesis that hypoxia-induced proliferation of fibroblasts would be dependent on distinct (compared with serum) activation and utilization patterns of mitogen-activated protein (MAP) kinases initiated by G␣ i/o proteins. We found that hypoxia stimulated increases in DNA synthesis and growth of quiescent fibroblasts in the absence of exogenous mitogens and also markedly augmented serum-stimulated growth responses. Hypoxia caused a transient activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), the time course and pattern of which was somewhat similar to that induced by serum but which was of lesser magnitude. On the other hand, hypoxia-induced activation of p38 MAP kinase was biphasic, whereas serum-stimulated activation of p38 MAP kinase was transient, and the magnitude of activation was greater for hypoxia compared with that of serum stimulation. ERK1/2, JNK1, and p38 MAP kinase but not JNK2 were necessary for hypoxia-induced proliferation because PD98059, SB202190, and JNK1 antisense oligonucleotides nearly ablated the growth response. JNK2 appeared to act as a negative modulator of hypoxia-induced growth because JNK2 antisense oligonucleotides led to an increase in DNA synthesis. In serum-stimulated cells, antisense JNK1 oligonucleotides and PD98059 had inhibitory effects on proliferation, whereas SB202190 led to an increase in DNA synthesis. Pertussis toxin, which blocks G␣ i/o -mediated signaling, markedly attenuated hypoxiainduced DNA synthesis and activation of ERK and JNK but not p38 MAP kinase. We conclude that hypoxia itself can act as a growth promoting stimulus for subsets of bovine neonatal adventitial fibroblasts largely through G␣ i/o -mediated activation of a complex network of MAP kinases whose specific contributions to hypoxia-induced proliferation differ from traditional serum-induced growth signals.

Enhanced proliferation of adventitial fibroblasts is a major contributor to the structural remodeling of the pulmonary artery (PA) that occurs during hypoxia-induced pulmonary hypertension. The mechanisms responsible for the exuberant growth of fibroblasts are unknown; however, protein kinase C (PKC) isozymes have previously been shown to be important in the enhanced growth properties of immature PA fibroblasts. We tested the hypotheses that PA adventitial fibroblasts from neonatal calves exposed chronically to hypoxia after birth would express augmented growth responses compared with fibroblasts from the control adventitia and that these properties would be associated with selective changes in expression of PKC isozymes. We studied the effects of serum, purified mitogens, and hypoxia on the growth of aggregate populations of fibroblasts isolated from the PA of neonatal control calves (Neo-C) and calves chronically exposed to hypoxia for 2 wk beginning on Day 1 of life (Neo-Hyp). Neo-Hyp fibroblasts demonstrated higher proliferative capabilities than did Neo-C cells in response to all the stimuli tested. Importantly, hypoxia was found to act synergistically with peptide mitogens (platelet-derived growth factor, basic fibroblast growth factor, insulin-like growth factor-I) to stimulate growth in Neo-Hyp but not in Neo-C cells. Using PKC-isozyme nonselective and selective inhibitors and immunoblot analysis, we found differences in utilization of PKC isozymes in Neo-Hyp and Neo-C fibroblasts and have identified PKC-betaI and -zeta as key contributors to the augmented growth of Neo-Hyp fibroblasts. Although the activity of PKC-betaI and -zeta isozymes was increased by hypoxia in serum-deprived Neo-C and Neo-Hyp fibroblasts, under normoxia, quiescent Neo-Hyp fibroblasts had higher PKC-zeta-specific activity than did Neo-C cells. These results suggest that neonatal PA adventitial fibroblasts acquire new growth properties in the setting of hypoxia- induced pulmonary hypertension and that the augmented proliferative characteristics of the Neo-Hyp fibroblasts might be associated with changes in specifc PKC isozyme expression and activation patterns.

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