Biphasic Erk1/2 activation sequentially involving Gs and Gi signaling is required in beta3-adrenergic receptor-induced primary smooth muscle cell proliferation

Abstract

The beta3 adrenergic receptor (B3-AR) reportedly induces cell proliferation, but the signaling pathways that were proposed, involving either Gs or Gi coupling, remain controversial. To further investigate the role of G protein coupling in B3-AR induced proliferation, we stimulated primary human myometrial smooth muscle cells with SAR150640 (B3-AR agonist) in the absence or presence of variable G-protein inhibitors. Specific B3-AR stimulation led to an Erk1/2 induced proliferation. We observed that the proliferative effects of B3-AR require two Erk1/2 activation peaks (the first after 3min, the second at 8h). Erk1/2 activation at 3min was mimicked by forskolin (adenylyl-cyclase activator), and was resistant to pertussis toxin (Gi inhibitor), suggesting a Gs protein signaling. This first signaling also required the downstream Gs signaling effectors PKA and Src. However, Erk1/2 activation at 8h turned out to be pertussis toxin-dependent, and PKA-independent, indicating a Gi signaling pathway in which Src and PI3K were required. The pharmacological inhibition of both the Gs and Gi pathway abolished B3-AR-induced proliferation. Altogether, these data indicate that B3-AR-induced proliferation depends on the biphasic activation of Erk1/2 sequentially induced by the Gs/PKA/Src and Gi/Src/PI3K signaling pathways.

Dose‐dependent biphasic leptin‐induced proliferation is caused by non‐specific IL‐6/NF‐κB pathway activation in human myometrial cells

“…This pathway has been widely studied as it is thought to be activated by virtually all mitogenic factors (Yang et al ., ). ERK1/2 is indeed a key regulator of the early cell cycle induction, as it is involved in G0/G1 and G1/S transition and our team demonstrated a key role for the biphasic transient activation of ERK1/2 in mymetrial cell proliferation (Hadi et al ., ). However, we only observed ERK1/2 phosphorylation and nuclear translocation in response to the lower dose of leptin.…”

Agonist‐induced desensitisation of β3‐adrenoceptors: Where, when, and how?

“…An alternative only rarely used in desensitisation studies are FRET-or BRET-based cAMP probes (Barak et al, 2008), but this has been used in only one study in the field (Milano et al, 2018). Another complication of AC and cAMP accumulation assays is that β 3 -adrenoceptors not only couple to G s proteins to stimulate AC but can also couple to G i proteins to inhibit it (Germack & Dickenson, 2006;Hadi et al, 2013;Sato et al, 2012;Soeder et al, 1999). Thus, unless G i proteins are inhibited, for instance, by pretreatment with pertussis toxin, AC and cAMP accumulation assays reflect a net effect of stimulation via G s and inhibition via G i ; in some models, this net effect may be an inhibition rather than a stimulation of cAMP accumulation (Germack & Dickenson, 2006).…”

β3-Adrenergic receptor-dependent modulation of the medium afterhyperpolarization in rat hippocampal CA1 pyramidal neurons

“…The ␤ 3adrenergic receptor signaling pathway is ill defined, with the receptor being reported to couple to G␣ s or G␣ i (Collins 2012). Other studies report that ␤ 3 -adrenergic receptors signal in a biphasic manner by coupling to both G␣ s and G␣ i (Bégin-Heick 1995;Gauthier et al 1996;Hadi et al 2013). Regardless of possible second messenger pathways, activation of ␤ 3 -adrenergic receptors causes relaxation of rat bladder, an effect blocked by ZD7288.…”