Loss of Gsα impairs liver regeneration through a defect in the crosstalk between cAMP and growth factor signaling

doi:10.1016/j.jhep.2015.08.036

Journal of Hepatology

Volume 64, Issue 2, February 2016, Pages 342-351

https://doi.org/10.1016/j.jhep.2015.08.036 Get rights and content

Background & Aims

The stimulatory G protein α subunit (G_sα) activates the cAMP-dependent pathway by stimulating the production of cAMP and participates in diverse cell processes. Aberrant expression of G_sα results in various pathophysiological disorders, including tumorigenesis, but little is known about its role in liver regeneration.

Methods

We generated a hepatocyte-specific G_sα gene knockout mouse to demonstrate the essential role of G_sα in liver regeneration using a mouse model with 70% partial hepatectomy (PH) or an intraperitoneal injection of carbon tetrachloride (CCl₄).

Results

G_sα inactivation dramatically impaired liver regeneration and blocked proliferating hepatocytes in G1/S transition due to the simultaneous depression of cyclin-dependent kinase 2 (CDK2) and cyclin E1. Loss of G_sα led to a fundamental alteration in gene profiles. Among the altered signaling cascades, the MAPK/Erk pathway, which is downstream of growth factor signaling, was disrupted secondary to a defect in phosphorylated Raf1 (pRaf1), resulting in a deficiency in phosphorylated CREB (pCREB) and CDK2 ablation. The lack of pRaf1 also resulted in a failure to phosphorylate retinoblastoma, which releases and activates E2F1, and a decrease in cyclin E1. Although these factors could be phosphorylated through both G_sα and growth factor signaling, the unique function of Raf1 in the growth factor cascade collapsed in response to the lack of G_sα.

Conclusion

The growth factor signaling pathway that promotes hepatocyte proliferation is dependent on G_sα signaling. Loss of G_sα leads to a breakdown of the crosstalk between cAMP and growth factor signaling and dramatically impairs liver regeneration.

Graphical abstract

Introduction

The liver has a unique capacity to rapidly and completely regenerate after a partial hepatectomy (PH) or chemical injury [1]. Following a two-thirds (or 70%) PH in rodents, quiescent hepatocytes synchronously re-enter the cell cycle and divide until they restore the original liver mass over a period of approximately 7 days. Three types of transmembrane receptors transmit extracellular mitotic signals, including ion channel-linked receptors, enzyme-linked receptors, and G protein-coupled receptors (GPCRs). Ion channel-linked receptors are mainly involved in rapid signaling events in electrically excitable cells, such as neurons. The roles of enzyme-linked receptor (including growth factor receptors and cytokine receptors) cascades that regulate liver regeneration have been comprehensively explored; however, little is known about the function of GPCRs in the regenerating liver.

Guanine nucleotide-binding proteins (G proteins) comprise a family of proteins that are involved in the transmission of GPCR-related signals from a variety of external stimuli to the interior of the cell [2]. There are two classes of G proteins: the first class functions as monomeric small GTPases, whereas the second class participates in a heterotrimeric G protein complex of α, β, and γ subunits. This protein complex functions as a molecular switch, in that when the heterotrimeric complex combines with a ligand, GDP is replaced by GTP and is released from the Gα subunit. This process is followed by the dissociation of Gα from Gβγ [3]. The stimulatory G protein α subunit (G_sα) activates the cAMP-dependent pathway via the stimulation of the production of cAMP from ATP [4]. cAMP then acts as a second messenger that interacts with and activates protein kinase A (PKA) [5]. PKA can phosphorylate countless downstream targets that are involved in a number of pathways.

The G_sα gene (GNAS in humans and Gnas in mice) is a complex imprinted gene that encodes multiple gene products through the use of alternative promoters and its first exon. Accumulating evidence has demonstrated that aberrant expression of G_sα leads to various dysfunctions in cell growth, proliferation, apoptosis, differentiation, and metabolism. Liver-specific disruption of G_sα increases hepatic glycogen synthesis and reduces the expression of enzymes involved in gluconeogenesis [6]. Mice with β-cell-specific G_sα deficiency develop severe early-onset, insulin-deficient diabetes accompanied by a severe defect in β-cell proliferation [7]. Recent studies have indicated that mutations in GNAS or G protein dysfunction are related to many diseases [8]. GNAS mutations are involved in the tumorigenesis of hepatobiliary and pancreatic tissues originating from the foregut endoderm [9]. Activating point mutations at codon 201 of GNAS have been detected in approximately two-thirds of intraductal papillary mucinous neoplasms and in half of intraductal papillary neoplasms of the bile duct [10], [11]. Frequent GNAS mutations have also been identified in intrahepatic cholangiocarcinomas and are associated with poor overall survival [11]. Although often absent in hepatocellular carcinoma, somatic GNAS-activating mutations are involved in the molecular pathway of hepatocellular adenomas by activating the IL6/STAT3 cascade [12]. G_sα seems to be closely involved in the regulation of cell proliferation, but the signaling pathway remains unclear. In the present study, we used a liver-specific G_sα knockout mouse model to define the biological function of G_sα in liver regeneration.

Section snippets

Mice

These experiments were approved by the Animal Care and Use Committee of Sichuan University. G_sα^loxP/loxP mice were kindly provided by Dr. HS Li, West China Second University Hospital, Sichuan University. Albumin-Cre transgenic mice were purchased from Cyagen Biosciences Inc., Guangzhou, China. Hepatic-G_sα^−/− mice were generated by crossing G_sα^loxP/loxP mice to albumin-Cre mice, and their genotypes were determined using PCR amplification of tail DNA (the primers for PCR are listed in

Impaired liver regeneration in G_sα^−/− mice following 70% PH or CCl₄ challenge

The hepatic-G_sα^−/− mice presented normal survival, body weight, food intake, and metabolic rates, consistent with a previous work by Chen M and colleagues [6]. The mutant livers were much larger in size and displayed greater glycogen deposition than their wild-type (WT) littermates (Supplementary Fig. 1).

To investigate the role of G_sα in liver regeneration, we performed a 70% PH on G_sα^−/− and WT mice. To our surprise, approximately 65% of the G_sα^−/− mice died within 30 h of surgery (Fig. 1A),

Discussion

Recent studies have indicated that mutations causing GNAS or G_sα dysfunction are related to various diseases that are characterized by aberrant cell proliferation [8], [11], [12], [26]. However, the signal cascades by which G_sα regulates cell proliferation are still not fully understood. In this study, we investigated the essential role of G_sα in liver regeneration and demonstrated that loss of G_sα robustly impairs hepatocyte proliferation.

Generally, 70% hepatectomy is a well-tolerated

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Acknowledgements

The authors thank Professor Huashun Li for kindly providing the G_sα^loxP/loxP mice. Microarray experiments were performed by KangChen Bio-Tech, Shanghai, China. This work was supported by grants from the National Key Clinical Project, the National Sciences and Technology Major Project of China (2012ZX10002-017), and the Science Fund for Outstanding Young Scholars of Sichuan University (2013SCU04B11).

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Taken together, our results reveal that Gsα plays a protective role in the development of AAA by inhibiting the HuR/KLF4-mediated SMC phenotype switch. Increasing evidence indicates that Gsα plays important roles in cell proliferation, apoptosis, differentiation and metabolism [23,24]. To detect the functions of Gsα in vivo, many tissue-specific Gsα knockout mice have been generated.
Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease without an effective pharmaceutical treatment. Genetic studies have proved the involvement of smooth muscle phenotype switch in the development of AAA. The alpha subunit of the heterotrimeric G stimulatory protein (Gsα) mediates receptor-stimulated production of cyclic adenosine monophosphate (cAMP). However, the role of smooth muscle Gsα in AAA formation remains unknown.
In this study, mice with knockout of smooth muscle-specific Gsα (Gsα^SMKO) were generated by cross-breeding Gsα^flox/flox mice with SM22-CreER^T2 transgenic mice, induced in adult mice by tamoxifen treatment. Gsα deficiency induced a smooth muscle phenotype switch from a contractile to a synthetic state. Mechanically, Gsα deletion reduced cAMP level and increased the level of human antigen R (HuR), which binds with the adenylate uridylate–rich elements of the 3′ untranslated region of Krüppel-like factor 4 (KLF4) mRNA, thereby increasing the stability of KLF4. Moreover, genetic knockdown of HuR or KLF4 rescued the phenotype switch in Gsα-deficient smooth muscle cells. Furthermore, with acute infusion of angiotensin II, the incidence of AAA was markedly higher in ApoE^−/−/Gsα^SMKO than ApoE^−/−/Gsα^flox/flox mice and induced increased elastic lamina degradation and aortic expansion. Finally, the levels of Gsα and SM α-actin were significantly lower while those of HuR and KLF4 were higher in human AAA samples than adjacent nonaneurysmal aortic sections.
Gsα may play a protective role in AAA formation by regulating the smooth muscle phenotype switch and could be a potential therapeutic target for AAA disease.
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Interestingly, several nonrecurrent alterations have been shown to affect other genes involved in cyclic adenosine 3′:5′ monophosphate (cAMP) signaling.6,7 The G protein α subunit activates the cAMP-dependent pathway by stimulating production of cAMP, and participates in diverse cell processes that cause various pathophysiologic disorders, including tumorigenesis.8 Altered cell cycle genes, selective oncoprotein activation, or loss of suppressor factors may be associated with aberrant growth factor signaling in pituitary adenomas.9
The present study was conducted to evaluate the levels of Cdk2, cyclin E, p21, and p27 in growth hormone adenomas (GHPAs) and analyze their association with clinicopathologic features.
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The levels of cyclin E and Cdk2 were much greater in the invasive group than in the noninvasive group (P < 0.05). Significant differences were found between cyclin E and p21 and tumor size (P < 0.05) and between cyclin E expression and invasion (P < 0.05). Tumors were more likely to require whole resection in patients with low cyclin E expression (P < 0.05). The high-level p27 group had better progression-free survival than did the low-level p27 group (P < 0.01). Quantitative real-time polymerase chain reaction and Western blot analysis revealed a similar trend for Cdk2, cyclin E, p21, and p27 protein levels in growth hormone specimens (P < 0.01). An average of 33 CpG sites per sample were analyzed using matrix-assisted laser desorption ionization time-of-flight mass array, and in 7 of the 33 CpG sites, the methylation levels of p27 were >50%. Statistically significant differences were found in 4 CpG sites between the invasive and noninvasive specimens (P < 0.01).
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The stimulatory G protein a subunit (G_sα) plays important roles in diverse cell processes including tumorigenesis. Activating mutations in G_sα gene (GNAS) have been reported to be associated with poor prognosis in various human carcinomas. Furthermore, G_sα signaling is crucial in promoting liver regeneration by interacting with growth factor signaling, indicating that G_sα might play a promoting role in cancer development. However, little is known about the correlation between G_sα levels and clinicopathological parameters in intrahepatic cholangiocarcinoma (ICC).
We performed immunoblotting to examine the expression levels of G_sα and Ki67 proteins in tumor tissues and the corresponding adjacent tissues. A total of 74 pair of specimens resected from 74 ICC patients were examined. The association between G_sα levels and clinicopathological findings and prognosis of the patients was evaluated.
Western blotting demonstrated that the expression of G_sα was significantly higher in ICC tissues compared with that in their corresponding adjacent tissues. G_sα protein was highly expressed in about half of ICC tissues (48.6%, 36/74) while only 28.4% (21/74) of tumor adjacent tissues showed G_sα high expression (P=0.011). High G_sα expression in ICC was significantly associated with the numbers of tumor nodules (P=0.037) and lymph node metastases (P=0.010). Moreover, the level of G_sα was significantly and positively correlated with Ki67 expression (P<0.001). In addition, the recurrence-free survival rate and overall survival rate in the G_sα high group were significantly lower than those in the G_sα low group (P=0.004 and P=0.005, respectively).
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Disruption of Gnas in smooth muscle of mice reduced intestinal motility and led to death within 4 weeks. Tamoxifen-induced disruption of Gnas in adult mice impaired contraction of intestinal smooth muscle and peristalsis. More than 80% of these died within 3 months of tamoxifen exposure, with features of intestinal pseudo-obstruction characterized by chronic intestinal dilation and dysmotility. Gsa deficiency reduced intestinal levels of cyclic adenosine monophosphate and transcriptional activity of the cyclic adenosine monophosphate response element binding protein 1 (CREB1); this resulted in decreased expression of the forkhead box F1 gene (Foxf1) and protein, and contractile proteins, such as myosin heavy chain 11; actin, α2, smooth muscle, aorta; calponin 1; and myosin light chain kinase. We found decreased levels of Gsa, FOXF1, CREB1, and phosphorylated CREB1 proteins in intestinal muscle layers of patients with chronic intestinal pseudo-obstruction, compared with tissues from controls.
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^†: The authors contributed equally to this work.

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Research ArticleLoss of Gsα impairs liver regeneration through a defect in the crosstalk between cAMP and growth factor signaling

Background & Aims

Methods

Results

Conclusion

Graphical abstract

Introduction

Section snippets

Mice

Impaired liver regeneration in Gsα−/− mice following 70% PH or CCl4 challenge

Discussion

Conflict of interest

Acknowledgements

Trends Biochem Sci

Neoplasia

J Hepatol

C R Acad Sci III

Kidney Int

Cell

Gastroenterology

Am J Pathol

J Hepatol

Cell

Liver regeneration

Hepatology

Genomic characterization of the human heterotrimeric G protein alpha, beta, and gamma subunit genes

DNA Res

The heterotrimeric G-protein GanB(alpha)-SfaD(beta)-GpgA(gamma) is a carbon source sensor involved in early cAMP-dependent germination in Aspergillusnidulans

Genetics

CAMP activation of PKA defines an ancient signaling mechanism

Proc Natl Acad Sci U S A

Increased glucose tolerance and reduced adiposity in the absence of fasting hypoglycemia in mice with liver-specific Gs alpha deficiency

J Clin Invest

Beta cell-specific deficiency of the stimulatory G protein alpha-subunit Gs alpha leads to reduced beta cell mass and insulin-deficient diabetes

Proc Natl Acad Sci U S A

Hepatobiliary and pancreatic neoplasms in patients with McCune-Albright syndrome

J Clin Endocrinol Metab

Whole-exome sequencing uncovers frequent GNAS mutations in intraductal papillary mucinous neoplasms of the pancreas

Sci Rep

GNAS and KRAS mutations are common in intraductal papillary neoplasms of the bile duct

PLoS One

Research Article
Loss of G_sα impairs liver regeneration through a defect in the crosstalk between cAMP and growth factor signaling

Impaired liver regeneration in G_sα^−/− mice following 70% PH or CCl₄ challenge