Published as doi: 10.1096/fj.05-5622fje.
(The FASEB Journal. 2006;20:1558-1560.)
© 2006 FASEB
Cooperative mitogenic signaling by G protein-coupled receptors and growth factors dependent on Gq/11
Kok Choi Kong*,
Charlotte K. Billington*,
Uma Gandhi*,
Reynold A. Panettieri, Jr.
and
Raymond B. Penn*,1
* Department of Internal Medicine and Center for Human Genomics, Wake Forest University Health Sciences, Winston-Salem, North Carolina, USA; and
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
1Correspondence: Wake Forest University Health Sciences Center, Center for Human Genomics, Medical Center Blvd., Winston Salem, NC 27157, USA. E-mail: rpenn{at}wfubmc.edu
SPECIFIC AIMS
This study aimed to: 1) identify the specific heterotrimeric G protein(s) that mediates cooperative mitogenic signaling by G protein-coupled receptors (GPCRs) and growth factors in human airway smooth muscle (ASM) cells; and 2) establish that such signaling was a robust phenomenon employed by multiple GPCRs.
PRINCIPAL FINDINGS
1. Thrombin augmentation of epidermal growth factor (EGF)-stimulated PI3K and p70S6K activity and [3H] thymidine incorporation is dependent on Gq/11 and not Gi/o or G12/13
Previous studies established that costimulation of human ASM cells with EGF plus thrombin results in additive or greater-than effects on Akt phosphorylation (which serves as a readout for PI3K activity), p70S6K phosphorylation (which correlates with p70S6K activity), and [3H]-thymidine incorporation. Heterologous expression of RGS proteins in human ASM cultures, induced by retroviral infection and selection for stable expression, or pretreatment with pertussis toxin was used to assess those heterotrimeric G proteins involved in these cooperative signaling and functional endpoints. Expression of GRK2NT-GFP, which functions as an RGS protein for Gq/11, significantly inhibited the increase in both the pS473Akt and pT389p70S6K levels, whereas expression of p115RhoGEFRGS-GFP (an RGS for G12/13) or pretreatment with pertussis toxin (inactivating Gi/o) had only minimal effects (Fig. 1
). In parallel experiments, GRK2NT-GFP but not p115RhoGEFRGS-GFP or pertussis toxin pretreatment significantly inhibited thrombin-mediated augmentation of EGF-stimulated [3H]-thymidine incorporation. Inhibition of Rho (with C3 exoenzyme) or Rho kinase (with Y27632), both downstream effectors of G12/13, also failed to inhibit the cooperative effects of thrombin of mitogenic signaling and DNA synthesis.
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Figure 1. Effects of RGS protein expression and Rho kinase inhibition on p70S6K, Akt phosphorylation, and ASM [3H]-thymidine incorporation stimulated by EGF and thrombin. ASM cells infected with GFP or GRK2NT-GFP (A, C, D, F) or GFP or p115RhoGEFRGS-GFP (B, C, D, F) were growth-arrested then stimulated with 10 ng/ml of EGF (E), 1 U/ml of thrombin (T), or both (E+T) for 30 min or 4 h. In (E) naive ASM cells were pretreated with Rho kinase inhibitor Y27623 (10µM) for 30 min prior to stimulation. Levels of phospho-p70S6K (T389), phospho-Akt (S473) and total Akt in cell lysates were assessed by immunoblotting. Representative blots (A, B, and E) and graphs depicting mean ± SE values of quantified phosphoprotein bands (C and D, n =5–7) are presented. Raw values for the 30 min EGF-stimulated condition were set to a value of 1.0 and all other values normalized accordingly. F) [3H]-thymidine incorporation stimulated by EGF, thrombin, or both in GFP-, GRK2NT-GFP-, or p115RhoGEFRGS-GFP-expressing lines (mean ±SE values, n =7 paired observations for GFP vs. GRK2NT-GFP, and n =4 paired observations for GFP vs. p115 RhoGEFRGS-GFP). *P <0.05, GFP vs. GRK2NT-GFP or p115 RhoGEFRGS-GFP group value for the indicated stimulatory condition.
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2. Both histamine and thromboxane also augment EGF-stimulated PI3K and p70S6K activity and [3H] thymidine incorporation, through a mechanism that involves Gßbeta;
subunits and Gq/11
The demonstrated requirement for Gq/11 in cooperative effects of thrombin on ASM mitogenic signaling and DNA synthesis led us to explore whether other Gq/11-coupled receptors in ASM used a similar mechanism to effect mitogenic signaling. In naïve ASM cultures, both histamine and U46619 (a stable thromboxane analog) increased late-phase EGF-stimulated pS473Akt and pT389p70S6K levels. As with costimulation with thrombin, costimulation with histamine or U46619 did not augment early-phase (30 min) activation of Akt and p70S6K by EGF. In cultures expressing one of two different Gßbeta; sequestrants (GRK2CT-GFP or G
iG203A), the increases in late-phase levels of pS473Akt and pT389p70S6K promoted by costimulation with histamine or U46619 were significantly inhibited, as were associated increases in EGF-stimulated [3H]-thymidine incorporation. As was the case with thrombin, expression of GRK2NT-GFP but not p115RhoGEFRGS-GFP or pertussis toxin pretreatment significantly inhibited the cooperative effects of histamine and U46619 on EGF-stimulated Akt and p70S6K phosphorylation and thymidine incorporation (Fig. 2
).
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Figure 2. Effects of GRK2CT-GFP, GiG203A, and GRK2NT-GFP expression on cooperative effects of EGF and histamine or U46619 in ASM cells. A) ASM cells stably expressing GFP, GRK2CT-GFP, GiG203A, or GRK2NT-GFP by retroviral infection were growth arrested before stimulated with 10 ng/ml EGF (E), 10 µM histamine (H), or both (E+H), 100 nM U46619 (U) or both EGF (E+U) for 6 h. Levels of phosphorylation of Akt at residue S473 and p70S6K at residue T389 were assessed by immunoblotting as described in Materials and Methods. Blots shown (A) are representative results obtained in six separate sets of lines derived from distinct donors and graphs depicting mean ± SE of quantified phosphoprotein bands (B) are presented. C) [3H]-thymidine incorporation stimulated by EGF, histamine, or both, U46619 or both EGF and U46619 in GFP-, GRK2CT-GFP-, GiG203A-GFP-, or GRK2NT-GFP-expressing lines (mean ±SE, n=5–6). *P<0.05, GFP vs. GRK2CT-GFP, GiG203A, or GRK2NT-GFP group value for the indicated stimulatory condition.
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3. Neither Src nor arrestins appear involved in mediating late-phase Akt or p70S6K phoshorylation by GPCRs
Because both Src and arrestin proteins have been shown to promote mitogenic signaling by GPCRs in various cell systems, we examined the effects of Src inhibition (using PP1) and arrestin overexpression/knockdown on thrombin, histamine, and U46619-mediated phosphorylation of Akt and p70S6K in the presence and absence of EGF. Pretreatment of ASM cells with PP1 did not influence the ability of any of the GPCR agonists to augment late-phase EGF-stimulated pS473Akt and pT389p70S6K levels. Similarly, overexpression of either arrestin2 or arrestin3 as GFP chimeras also failed to affect the cooperative effects of thrombin, histamine, or U46619. Stable expression of siRNA targeting arrestin2/3, resulting in a 50–60% reduction in arrestin2/3 protein expression, also had no effect.
CONCLUSIONS AND SIGNIFICANCE
Increased ASM mass is recognized as a principal feature of airway remodeling that is characteristic of chronic asthma and contributes to both fixed airflow obstruction and exaggerated response to contractile agents. Given the limitations of in vivo models capable of examining this phenomenon, limited insight exists into the mechanisms mediating ASM hyperplasia and hypertrophy. However, it is appreciated that levels of both growth factors and GPCR agonists can be elevated in the asthmatic airway and that various combinations of growth factors and GPCRs agonists can synergistically stimulate the proliferation of ASM cells in culture. Our initial studies characterizing the mitogenic signaling associated with GPCR-mediated augmentation of EGF-stimulated ASM growth demonstrated an association with prolonged, increased activation of p70S6K, and not of p42/p44 MAPK. Our more recent study demonstrated that thrombin, which activates protease-activated receptors in ASM, augments EGF-stimulated PI3K and p70S6K activity, as well as thymidine incorporation, via a mechanism dependent on Gßbeta; subunits. GPCR-mediated regulation of p70S6K activity involved specific regulation of T389 phosphorylation, as specific inhibition of p42/p44 and the associated phosphorylation of the proline-directed residues S421/T424 did not affect p70S6K activity or the cooperative effect on ASM DNA synthesis.
The present study offers two major findings. First, Gq/11 is identified as the principal G protein mediating the cooperative effects of thrombin on EGF-induced late-phase PI3K/p70S6K activation and growth in ASM in a Gßbeta;-dependent manner. The finding that Gq/11, but not Gi/o, is the principal G protein responsible for the mechanism is rather intriguing as Gi/o tends to be the most abundantly expressed heterotrimeric G protein in non-neuronal cells, including human ASM and is, therefore, the most likely source of Gßbeta;. Second, we demonstrate that two other GPCRs, namely the Gq/11-coupled H1 histamine receptor, and the T Prostanoid (TP, or TXA2) receptors, which can couple to Gq/11, Gi/o, and G12/13, also exhibit similar cooperative mitogenic effects with EGF via a similar mechanism. Consistent with this finding, others have recently shown that augmented growth of bovine ASM cells induced by platelet-derived growth factor and methacholine is mediated through m3- (Gq/11-coupled) and not the m2- (Gi/o-coupled) muscarinic acetylcholine receptors. Collectively, these findings strongly suggest that cooperative mitogenic signaling by GPCRs and receptor tyrosine kinases is a robust phenomenon that uses the fundamental mechanism of Gq/11-derived Gßbeta; subunits promoting late-phase PI3K and p70S6K activity.
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Figure 3. Schematic representation of cooperative activation of PI3K and p70S6K by receptor tyrosine kinase and GPCRs, leading to increased ASM proliferation. Agonists (histamine, thromboxane, or thrombin) activating Gq-coupled receptors enhance EGF receptor-activated PI3K activity via release of Gßbeta; subunits, resulting in increased activation of downstream targets Akt and p70S6K, which in turn increases ASM proliferation.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5622fje
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Copyright © 2006 by The Federation of American Societies for Experimental Biology.
