To identify the signaling pathway that mediates the adrenergic stimulation of the expression of the gene for vascular endothelial growth factor (VEGF) during physiologically induced angiogenesis, we examined mouse brown adipocytes in primary culture. The endogenous adrenergic neurotransmitter norepinephrine (NE) induced VEGF expression 3-fold, in a dose-and time-dependent manner (EC 50 Ϸ 90 nM). Also, the hypoxia-mimicking agent cobalt, as well as serum and phorbol ester, induced VEGF expression, but the effect of NE was additive to each of these factors, implying that a separate signaling mechanism for the NE-mediated induction was activated. The NE effect was abolished by propranolol and mimicked by isoprenaline or BRL-37344 and was thus mediated via ␤-adrenoreceptors. The NE-induced VEGF expression was fully cAMP mediated, an effect which was inhibited by H-89 and thus was dependent on protein kinase A activity. Involvement of other adrenergic signaling pathways (␣ 1 -adrenoreceptors, Ca 2؉ , protein kinase C, ␣ 2 -adrenoreceptors, and pertussis toxinsensitive G i -proteins) was excluded. The specific inhibitor of Src tyrosine kinases, PP2, markedly reduced the stimulation by NE, which demonstrates that a cAMP-dependent Src-mediated pathway is positively connected to VEGF expression. However, inhibition of Erk1/2 MAP kinases by PD98059 was without effect. NE did not prolong VEGF mRNA half-life and its effect was thus transcriptional, and was independent of protein synthesis. These results demonstrate that adrenergic stimulation, through ␤-adrenoreceptor/cAMP/protein kinase A signaling, recruits a pathway that branches off from the NE-activated Src-Erk1/2 cascade to enhance transcription of the VEGF gene.

A novel signaling pathway for mediation of ␤ 3 -adrenergic activation of the mitogen-activated protein kinases Erk1/2 (associated with proliferation, differentiation, and apoptosis) has recently been proposed, which implies mediation via constitutively coupled G i -proteins and G␤␥-subunits, distinct from the classical cAMP pathway of ␤-adrenergic stimulation. To verify the significance of this pathway in cells in primary cultures that entopically express ␤ 3 -adrenoreceptors, we examined the functionality of this pathway in cultured brown adipocytes. Norepinephrine activated Erk1/2 via both ␤ 3 receptors and ␣ 1 receptors but not via ␣ 2 receptors. Forskolin induced Erk1/2 activation similarly to ␤ 3 activation, indicating cAMP-mediation; this induction could be inhibited with H89, implying protein kinase A mediation. The G i -pathway was functional in these cells, as pertussis toxin increased agonist-induced cAMP accumulation. However, pertussis toxin was unable to affect adrenergically induced Erk1/2 activation. Also, wortmannin was without effect, implying that G␤␥ activation of the phosphatidylinositol 3-kinase pathway was not involved. PP1/2, which inhibits Src, abolished both ␤ 3 -and ␣ 1 -induced Erk1/2 activation. Thus, the proposed novel G i pathway for ␤ 3 mediation is not universal, because it is not functional in the untransformed primary cell culture system with entopically expressed ␤ 3 receptors examined here. Here, the ␤ 3 signal is mediated classically via cAMP/protein kinase A. ␤ 3 and ␣ 1 signals converge at Src, which thus mediates Erk1/2 activation in both pathways.

Brown adipose tissue hyperplasia is a fundamental response to low ambient temperature. We show here that cold exposure of an animal markedly increased the phosphorylation of mitogen-activated protein kinase (p42/p44) Erk1 and Erk2 in brown adipose tissue, and protected cells in the tissue from apoptosis. We also show that cessation of the sympathetic stimulus, by transferring cold-adapted animals to 28°C, caused an increased rate of apoptosis in the tissue. In primary cultures of brown adipose tissue, norepinephrine (NE) stimulated both the phosphorylation and the activity of Erk1/2 via the Erk kinase MEK, and protected the cells form apoptosis. Similarly, agonist stimulation of ␣ 1 -and ␤-adrenergic receptors and increases in the intracellular level of Ca 2؉ and cAMP stimulated the phosphorylation of Erk1/2. Agonist stimulation of ␣ 1 -and ␤-adrenergic receptors, and increased intracellular cAMP level also promoted the cell survival. Furthermore, NE stimulated the expression and secretion of basic fibroblast growth factor (bFGF), which further promoted the cell survival via MEK-dependent activation of Erk1/2. In essence, we show that Erk1/2 has a critical role in promoting NE-and bFGF-dependent survival of brown adipocytes, and propose that NE-and bFGF-dependent regulation of the cell survival is involved in the coldinduced hyperplasia of brown adipose tissue.

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