Regulation of the phosphatidylinositol 3-kinase,Akt/protein kinase B,FRAP/mammalian target of rapamycin,and ribosomal S6 kinase 1 signaling pathways by thyroid-stimulating hormone (TSH) and stimulating type TSH receptor antibodies in the thyroid gland

Thyroid-stimulating hormone (TSH) regulates the growth and differentiation of thyrocytes by activating the TSH receptor (TSHR). This study investigated the roles of the phosphatidylinositol 3-kinase (PI3K), PDK1, FRAP/mammalian target of rapamycin, and ribosomal S6 kinase 1 (S6K1) signaling mechanism by which TSH and the stimulating type TSHR antibodies regulate thyrocyte proliferation and the follicle activities in vitro and in vivo. The TSHR immunoprecipitates exhibited PI3K activity, which was higher in the cells treated with either TSH or 8-bromo-cAMP. TSH and cAMP increased the tyrosine phosphorylation of TSHR and the association between TSHR and the p85alpha regulatory subunit of PI3K. TSH induced a redistribution of PDK1 from the cytoplasm to the plasma membrane in the cells in a PI3K- and protein kinase A-dependent manner. TSH induced the PDK1-dependent phosphorylation of S6K1 but did not induce Akt/protein kinase B phosphorylation. The TSH-induced S6K1 phosphorylation was inhibited by a dominant negative p85alpha regulatory subunit or by the PI3K inhibitors wortmannin and LY294002. Rapamycin inhibited the phosphorylation of S6K1 in the cells treated with either TSH or 8-bromo-cAMP. The stimulating type TSHR antibodies from patients with Graves disease also induced S6K1 activation, whereas the blocking type TSHR antibodies from patients with primary myxedema inhibited TSH- but not the insulin-induced phosphorylation of S6K1. In addition, rapamycin treatment in vivo inhibited the TSH-stimulated thyroid follicle hyperplasia and follicle activity. These findings suggest an interaction between TSHR and PI3K, which is stimulated by TSH and cAMP and might involve the downstream S6K1 but not Akt/protein kinase B. This pathway may play a role in the TSH/stimulating type TSH receptor antibody-mediated thyrocyte proliferation in vitro and in the response to TSH in vivo.     

Induction of sodium/iodide symporter (NIS) expression and radioiodine uptake in non-thyroid cancer cells

Background

This study was designed to explore the therapeutic potential of suppressing MAP kinase and PI3K/Akt pathways and histone deacetylase (HDAC) to induce the expression of sodium/iodide symporter (NIS) and radioiodine uptake in non-thyroid cancer cells.

Methods

We tested the effects of the MEK inhibitor RDEA119, the Akt inhibitor perifosine, and the HDAC inhibitor SAHA on NIS expression in thirteen human cancer cell lines derived from melanoma, hepatic carcinoma, gastric carcinoma, colon carcinoma, breast carcinoma, and brain cancers. We also examined radioiodine uptake and histone acetylation at the NIS promoter in selected cells.

Results

Overall, the three inhibitors could induce NIS expression, to various extents, in melanoma and all the epithelial carcinoma-derived cells but not in brain cancer-derived cells. SAHA was most effective and its effect could be significantly enhanced by RDEA119 and perifosine. The expression of NIS, at both mRNA and protein levels, was most robust in the melanoma cell M14, hepatic carcinoma cell HepG2, and the gastric carcinoma cell MKN-7 cell. Radioiodine uptake was correspondingly induced, accompanied by robust increase in histone acetylation at the NIS promoter, in these cells when treated with the three inhibitors.

Conclusions

This is the first demonstration that simultaneously suppressing the MAP kinase and PI3K/Akt pathways and HDAC could induce robust NIS expression and radioiodine uptake in certain non-thyroid human cancer cells, providing novel therapeutic implications for adjunct radioiodine treatment of these cancers.     

Sp1 is involved in Akt-mediated induction of VEGF expression through an HIF-1-independent mechanism

Increased expression of vascular endothelial growth factor (VEGF) contributes to the growth of many tumors by increasing angiogenesis. Although hypoxia is a potent inducer of VEGF, we previously showed that epidermal growth factor receptor amplification and loss of PTEN, both of which can increase phosphatidylinositol-3-kinase (PI3K) activity, increase VEGF expression. Using both adenoviral vectors and a cell line permanently expressing constitutively active myristoylated Akt (myrAkt), we show that activation of Akt, which is downstream of PI3K, increases VEGF expression in vitro and increases angiogenesis in a Matrigel plug assay. Transient transfection experiments using reporter constructs containing the VEGF promoter showed that up-regulation of VEGF by Akt is mediated through Sp1 binding sites located in the proximal promoter. Small interfering RNA directed against Sp1 prevented the induction of VEGF mRNA in response to myrAkt but not to hypoxia. Expression of myrAkt is associated with increased phosphorylation of Sp1 and its increased binding to a probe corresponding to the -88/-66 promoter region. In conclusion, our results indicate that Sp1 is required for transactivation of the VEGF by Akt. Others have proposed that the PI3K/Akt pathway can increase VEGF expression via the hypoxia-inducible factor 1 (HIF-1); however, our results suggest an alternative mechanism can also operate.     

A novel signaling pathway for beta-adrenergic receptor-mediated activation of phosphoinositide 3-kinase in H9c2 cardiomyocytes

Stimulation of cardiac beta-adrenergic receptors (beta-AR) activates both the G(s)- and G(i)-coupled signaling cascades, including the phosphoinositide 3 kinase (PI3K) pathway, that have important physiological implications. Multiple isoforms of PI3K exist in the heart. The goals of this study were to examine the intracellular signaling pathways linking beta-AR to PI3K and to identify the PI3K isoform mediating this transactivation in a cardiac context. Acute beta-AR stimulation with isoproterenol resulted in increased tyrosine kinase-associated PI3K activity and phosphorylation of Akt and p70S6K in H9c2 cardiomyocytes. Cotreatment with ICI-118,551, but not CGP-20712, abolished the increase in PI3K activity, suggesting a beta(2)-AR-mediated event. PI3K activation was also abrogated by cotreatment with pertussis toxin, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2, a selective Src-family tyrosine kinases inhibitor), or AG-1296 [selective platelet-derived growth factor receptor (PDGFR) inhibitor] but not with an inhibitor for protein kinase A, protein kinase C, Ras, adenylyl cyclase, epidermal growth factor receptor, or insulin-like growth factor-1 receptor. beta-AR stimulation induced an increase in tyrosine phosphorylation of PDGFR, which was abolished by inhibition of Src either by PP2 or small interfering RNA. Moreover, H9c2 cardiomyocytes stably transfected with a vector expressing a Gbetagamma sequestrant peptide derived from the COOH-terminus of beta-AR kinase-1 failed to activate PI3K after beta-AR stimulation, suggesting Gbetagamma is required for the transactivation. Furthermore, acute beta-AR stimulation in vivo resulted in increases in PDGFR-associated PI3K and PI3Kalpha isoform activities but not the activities of other isoforms (PI3Kbeta, -delta, -gamma) in adult mouse heart. Taken together, these data provide in vitro and in vivo evidence for a novel mechanism of beta-AR-mediated transactivation of cardiac PI3Kalpha via sequential involvement of Galpha(i)/Gbetagamma, Src, and PDGFR.     

Dystrophin glycoprotein complex‐associated Gβγ subunits activate phosphatidylinositol‐3‐kinase/Akt signaling in skeletal muscle in a laminin‐dependent manner

Previously, we showed that laminin‐binding to the dystrophin glycoprotein complex (DGC) of skeletal muscle causes a heterotrimeric G‐protein (Gαβγ) to bind, changing the activation state of the Gsα subunit. Others have shown that laminin‐binding to the DGC also leads to Akt activation. Gβγ, released when Gsα is activated, is known to bind phosphatidylinositol‐3‐kinase (PI3K), which activates Akt in other cells. Here, we investigate whether muscle Akt activation results from Gβγ, using immunoprecipitation and immunoblotting, and purified Gβγ. In the presence of laminin, PI3K‐binding to the DGC increases and Akt becomes phosphorylated and activated (pAkt), and glycogen synthase kinase is phosphorylated. Antibodies, which specifically block laminin‐binding to α‐dystroglycan, prevent PI3K‐binding to the DGC. Purified bovine brain Gβγ also caused PI3K and Akt activation. These results show that DGC‐Gβγ is binding PI3K and activating pAkt in a laminin‐dependent manner. Mdx mice, which have greatly diminished amounts of DGC proteins, display elevated pAkt signaling and increased expression of integrin β1 compared to normal muscle. This integrin binds laminin, Gβγ, and PI3K. Collectively, these suggest that PI3K is an important target for the Gβγ, which normally binds to DGC syntrophin, and activates PI3K/Akt signaling. Disruption of the DGC in mdx mouse is causing dis‐regulation of the laminin‐DGC‐Gβγ‐PI3K‐Akt signaling and is likely to be important to the pathogenesis of muscular dystrophy. Upregulating integrin β1 expression and activating the PI3K/Akt pathway in muscular dystrophy may partially compensate for the loss of the DGC. The results suggest new therapeutic approaches to muscle disease. J. Cell. Physiol. 219: 402–414, 2009. © 2008 Wiley‐Liss, Inc.     

Role of guanine nucleotide exchange factor P-Rex-2b in sphingosine 1-phosphate-induced Rac1 activation and cell migration in endothelial cells

Endothelial cell (EC) migration has an important role in angiogenesis. Sphingosine-1 phosphate (S1P) stimulates EC migration via activation of Gi proteins. In this study, we characterized a mouse guanine nucleotide exchange factor (GEF) P-Rex2b for its regulation by Gbetagamma and PI3K and its role in S1P-induced Rac1 activation and cell migration in ECs. We found that co-expression of Gbetagamma or an active form of PI3K (PI3K(AC)) with P-Rex2b increased the SRE.Luciferase (SRE.L) reporter gene activity that can be stimulated by the Rho family of small GTPases including Rac1. Co-expression with P-Rex2b of Gbetagamma and PI3K(AC) or wild type PI3Kgamma that can be activated by Gbetagamma led to further increases in the reporter gene activity. Together with the finding that co-expression of Gbetagamma and/or PI3K(AC) increased the levels of active Rac1, we conclude that P-Rex2b is a Rac GEF that can be regulated by Gbetagamma and PI3K. Additionally, we demonstrated that Gbetagamma interacted with P-Rex2b, probably through P-Rex2b sequences at the PH domain and that the DEP and PDZ domains of P-Rex2b exerted an inhibitory effect on P-Rex2b's activity because their deletion increased the SER.L reporter gene activity. Furthermore, we found that P-Rex2b is involved in S1P-induced Rac1 activation and cell migration in ECs because siRNA-mediated suppression of P-Rex2b expression in ECs-diminished Rac1 activation and cell migration in response to S1P. Therefore, P-Rex2b is a physiologically significant Rac1 GEF that has an important role in the regulation of EC migration.     

TSH signaling and cell survival in 3T3-L1 preadipocytes

Thyroid-stimulating hormone (TSH) action in adipose tissue remains largely unknown. Our previous work indicates that human preadipocytes express functional TSH receptor (TSHR) protein, demonstrated by TSH activation of p70 S6 kinase (p70 S6K). We have now studied murine 3T3-L1 preadipocytes to further characterize TSH signaling and cellular action. Western blot analysis of 3T3-L1 preadipocyte lysate revealed the 100-kDa mature processed form of TSHR. TSH activated p70 S6K and protein kinase B (PKB/Akt), as measured by immunoblot analysis. Preincubation with wortmannin or LY-294002 completely blocked TSH activation of p70 S6K and PKB/Akt, implicating phosphoinositide 3-kinase (PI3K) in their regulation. TSH increased phosphotyrosine protein(s) in the 125-kDa region and augmented the associated PI3K activity fourfold. TSH had no effect on cAMP levels in 3T3-L1 preadipocytes, suggesting that adenylyl cyclase is not involved in TSH activation of the PI3K-PKB/Akt-p70 S6K pathway. 3T3-L1 preadipocyte cell death was reduced by 29-76% in serum-deprived (6 h) preadipocytes treated with 1-20 microM TSH. In the presence of 20 microM TSH, an 88% reduction in terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-positive cells was observed in serum-starved (3 h) 3T3-L1 preadipocytes as well as a 93% reduction in the level of cleaved activated caspase 3. In summary, TSH acts as a survival factor in 3T3-L1 preadipocytes. TSH does not stimulate cAMP accumulation in these cells but instead activates a PI3K-PKB/Akt-p70 S6K pathway.