Insulin receptor substrate-1-mediated enhancement of growth hormone-induced mitogen-activated protein kinase activation

Interaction of GH with the cell-surface GH receptor (GHR) causes activation of the GHR-associated tyrosine kinase, JAK2, and consequent triggering of signaling cascades including the STAT, Ras/Raf/MEK1/MAP kinase, and insulin receptor substrate-1(IRS-1)/PI3kinase pathways. We previously showed that IRS- and GHR-deficient 32D cells that stably express the rabbit GHR and rat IRS-1 (32D-rbGHR-IRS-1) exhibited markedly enhanced GH-induced proliferation and MAP kinase (ERK1 and ERK2) activation compared with cells expressing only the GHR (32D-rbGHR). We now examine biochemical mechanism(s) by which IRS-1 augments GH-induced MAP kinase activation. Time-course experiments revealed a similarly transient (maximal at 15 min) GH-induced ERK1 and ERK2 activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells, but, consistent with our prior findings, substantially greater activation was seen in the IRS-1-containing cells. In both cells, GH-induced MAP kinase activation was markedly blunted by the MEK1 inhibitor, PD98059, but not by the PKC inhibitor, GF109203X. Interestingly, pretreatment with the PI3K inhibitor, wortmannin (EC50 approximately 10 nM), significantly reduced GH-induced MAP kinase activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells. This same pattern in both cells of IRS-1-dependent augmentation and IRS-1-independent wortmannin sensitivity was also observed for GH-induced activation of Akt and MEK1 (using state-specific antibody blotting for both), despite the lack of difference in GHR, JAK2, SHP-2, p85, Akt, Ras, Raf-1, MEK1, ERK1, or ERK2 abundance between the two cells. A different PI3K inhibitor, LY294002 (50 microM), substantially inhibited (roughly 72%) GH-induced MAP kinase activation in 32D-rbGHR-IRS-1 cells, but only marginally (and statistically insignificantly) inhibited GH-induced MAP kinase activation in 32D-rbGHR cells. Because GH-induced Akt activation was completely inhibited in both cells by the same concentration of LY294002, these findings indicate that the wortmannin sensitivity of both the IRS-1-independent and -dependent GH-induced MAP kinase activation may reflect the activity of another wortmannin-sensitive target(s) in addition to PI3K in mediation of GH-induced MAP kinase activation in these cells. Notably, GH-induced STAT5 tyrosine phosphorylation, unlike Akt or MAPK activation, did not differ between the cells. Finally, while GH promoted accumulation of activated Ras in both cells, both basal and GH-induced activated Ras levels were greater in cells expressing IRS-1 than in 32D-rbGHR cells. These data indicate that while GH induces tyrosine phosphorylation of STAT5 and activation of the Ras/Raf/MEK1/MAPK and PI3K pathways, IRS-1 expression augments the latter two more than the former.     

Insulin-like growth factor-I-dependent signal transduction pathways leading to the induction of cell growth and differentiation of human neuroblastoma cell line SH-SY5Y: the roles of MAP kinase pathway and PI 3-kinase pathway

IGF-I regulates cell growth, differentiation, and survival in many cultured nerve cell lines. The present study was undertaken in the human neuroblastoma cell line, SH-SY5Y, to elucidate whether there are differences in the IGF-dependent signal transduction pathways that stimulate proliferation compared to those that induce differentiation. Quiescent SH-SY5Y cells were treated with IGF-I in the presence or absence of PD98059 (an inhibitor of MEK, a MAP kinase kinase) or LY294002 (an inhibitor of PI 3-kinase). Cell growth was assessed by measuring [3H]thymidine incorporation into DNA and cell number. Cell differentiation was assessed by measuring mRNA levels of NPY and neurite outgrowth. IGF-I both induced cell proliferation and differentiation. It stimulated tyrosine phosphorylation of the type I IGF receptor (IGF-IR) beta-subunit, IRS-I, IRS-2, and Shc, and these changes were associated with activation of Erk and Akt. PD98059 inhibited activation of Erk and LY294002 repressed activation of Akt in response to IGF-I, but did not affect tyrosine phosphorylation of the IGF-IR, IRS-1, IRS-2, or Shc. Each PD98059 and LY294002 inhibited IGF-I-dependent cell proliferation in a concentration-dependent manner. In contrast, each of these inhibitors only partially depressed NPY gene expression induced by IGF-I and slightly inhibited IGF-I-mediated neurite outgrowth; however, when both PD98059 and LY294002 were present, IGF-I-dependent NPY gene expression and neurite outgrowth were abolished completely. These results suggest that in these nerve cells, 1) the IGF-I signals through the MAP kinase pathway and PI-3 kinase pathway are independently essential to induce IGF-I-dependent growth, and 2) alternate activation of the MAP kinase pathway and PI 3-kinase pathway is sufficient for the cells to undergo IGF-I-dependent differentiation.     

Insulin-like growth factor signaling pathways in rat hepatic stellate cells: importance for deoxyribonucleic acid synthesis and hepatocyte growth factor production

It has been shown recently that insulin-like growth factor 1 (IGF-1) increases both DNA synthesis and hepatocyte growth factor (HGF) production in cultured hepatic stellate cells. In this study, we used selective blockers to investigate crucial signaling pathways for these effects of IGF-1 in cultured rat hepatic stellate cells. Both LY 294002 [a phosphatidylinositol 3-kinase (PI3-K) inhibitor], and rapamycin [a blocker of activation of the serine/threonine p70 S6 kinase (p70S6K), a molecule downstream from PI3-K] completely reversed the IGF-1-induced stimulation of DNA synthesis. Mitogen-activated protein kinase (MAPK) inhibition by PD 98059 had a less pronounced suppressory effect, although the used PD 98059 dose was fully effective in inhibiting MAPK phosphorylation. Both LY 294002 and PD 98059 lowered the IGF-1-induced increase of HGF in the medium by about 40%, but LY 294002 was 10 times more potent than PD 98059. Inhibition of p70S6K activation by rapamycin blocked IGF-1-induced DNA synthesis but not the increase in HGF. In conclusion, PI3-K (and, to some extent, MAPK) signaling pathways seem to be important for IGF-1-stimulated DNA synthesis and HGF production. DNA synthesis also seems to be dependent on rapamycin-sensitive activation of the PI3-K effector p70S6K.     

Participation of growth factor signal transduction pathways in estradiol facilitation of female reproductive behavior

Estradiol (E(2)) regulates female reproductive behavior (lordosis) by acting on estrogen-sensitive neurons. We recently showed that E(2) facilitation of lordosis behavior requires concurrent activation of brain IGF-I receptors. The present study confirmed this finding and sought to identify the downstream signaling pathways involved in estrogen/IGF-I priming of lordosis. Intracerebroventricular infusions of a selective IGF-I receptor antagonist were administered to ovariectomized rats every 12 h beginning 1 h before the first of two daily E(2) injections. IGF-I receptor blockade partially inhibits lordosis if the antagonist is infused throughout the 2-d estrogen treatment period but not if it is administered only during the first or last 12 h of estrogen treatment. Because E(2) and IGF-I can activate phosphatidylinositol-3-kinase (PI3K) and MAPK, we infused agents that block PI3K and/or MAPK activity as described above. Both PI3K inhibitors (wortmannin and LY294002) and MAPK inhibitors (PD98059 and U0126) partially attenuate lordosis when administered during estrogen priming. None of these drugs modifies lordosis if they are infused only once, during the last 12 h of estrogen treatment. When both wortmannin and PD98059 are infused during E(2) priming, lordosis behavior is completely abolished. These data suggest that activation of both PI3K and MAPK by E(2) and IGF-I mediates hormonal facilitation of lordosis behavior.     

Roles of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways in stimulation of vascular smooth muscle cell migration and deoxyriboncleic acid synthesis by insulin-like growth factor-I.

Insulin-like growth factor-I (IGF-I) is a potent stimulator of vascular smooth muscle cell (SMC) migration, a process that contributes to the accumulation of SMC within atherosclerotic lesions. Our previous studies have shown that IGF-I increases the affinity of the alphaVbeta3 integrin toward ligands and that occupancy of this integrin is indispensable for IGF-I to stimulate cell migration. In this study, the role of phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinase (MAPK) pathways in IGF-I induced cell motility and integrin activation was studied using porcine aortic smooth muscle cells (pSMC). Two structurally different inhibitors of PI 3-kinase decreased IGF-I-stimulated pSMC migration in a dose-dependent manner. The IC50 of wortmannin for inhibiting migration was 10 nM, and that of LY294002 was 0.3 microM. These inhibitors also suppressed IGF-I-induced phosphorylation of protein kinase B PKB/Akt at Ser437 using concentrations that also inhibited cell motility. PD98059, an inhibitor of the MAPK pathway, was somewhat less potent than PI 3-kinase inhibitors in blocking cell migration that had been stimulated by IGF-I. When IGF-I increased migration of pSMC 2.1-fold above control, 100 nM wortmannin inhibited this response by 79%, 1 microM LY294002 inhibited it by 58%, and 50 microM PD98059 caused a 34% reduction. In comparison, 100 nM wortmannin inhibited IGF-I stimulated DNA synthesis by 57%, 1 microM LY294002 inhibited it by 59%, whereas 50 microM PD98059 suppressed it completely. Thus, activation of PI 3-kinase plays the major role in IGF-I-stimulated migration and proliferation of pSMC. While the activation of the MAPK pathway seems to be necessary for stimulation of mitogenesis by IGF-I, the contribution of this pathway in IGF-I-induced cell migration is limited in pSMC. Interestingly, neither PI 3-kinase inhibitors nor PD98059 blocked the increase in alphaVbeta3 integrin affinity that followed IGF-I treatment. Therefore, although both the PI 3-kinase and MAPK pathways were used by IGF-I to increase migration of pSMC, alphaVbeta3 integrin activation did not depend on either PI 3-kinase or MAPK activation, suggesting the possible importance of some other signal transduction pathway to account for its full actions on pSMC.     

Role of phosphatidylinositol-3 kinase and its association with Gab1 in thrombopoietin-mediated up-regulation of platelet function

OBJECTIVE: Human blood platelets are easily available physiologic target cells for thrombopoietin (TPO). TPO up-regulates platelet aggregation and alpha-granule secretion induced by various agonists. We investigated the role of phosphatidylinositol 3-kinase (PI3K) and its association with Gab1 in TPO-mediated up-regulation of platelet function. MATERIALS AND METHODS: PI3K inhibitors (wortmannin and LY294002) and a MAP/ERK-kinase (MEK) inhibitor (PD98059) were used to investigate the role of these kinases in TPO-mediated up-regulation of platelet function. To elucidate the molecules associated with PI3K, we performed immunoprecipitation and pull-down experiments followed by immunoblotting. In vitro kinase assay also was performed to detect extracellular signal-regulated kinase (ERK) kinase activity. RESULTS: TPO up-regulated platelet alpha-granule secretion and aggregation induced by thrombin, which was dose-dependently inhibited by preincubation with wortmannin or LY294002. Immunoprecipitation and pull-down experiments revealed that regulatory subunit of PI3K, p85, was rapidly associated with tyrosine-phosphorylated Gab1 via its n- and c-terminal SH2 domains. Pretreatment of platelets with TPO dramatically augmented the thrombin-induced ERK activation, which was almost completely inhibited by LY294002. Furthermore, a MEK inhibitor, PD98059, not completely but significantly inhibited TPO-mediated up-regulation of thrombin-induced alpha-granule secretion. CONCLUSION: TPO induces the association of tyrosine-phosphorylated Gab1 with p85-PI3K. In downstream signaling, ERK is PI3K-dependently activated, which plays a critical role for TPO-mediated up-regulation of platelet function.     

Insulin-like growth factor-I receptor in proliferation and motility of pancreatic cancer

AIM:To develop a molecular therapy for pancreatic cancer, the insulin-like growth factor-I (IGF-I) signaling pathway was analyzed.METHODS: Pancreatic cancer cell lines (MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4) were cultured in media with 10 mL/L fetal bovine serum. Western blotting analysis was performed to clarify the expression of IGF-I receptor (IGF-IR). Picropodophyllin (PPP), a specific inhibitor of IGF-IR, LY294002, a specific inhibitor of phosphatidylinositol3 kinase (PI3K), and PD980...     

Resveratrol inhibits expression and binding activity of the monocyte chemotactic protein-1 receptor, CCR2, on THP-1 monocytes

Monocyte chemotactic protein-1 and its receptor, CCR2, play a key role in atherosclerosis. We determined the effect of the polyphenol, resveratrol, on CCR2 and the mechanisms involved. Resveratrol treatment inhibited 125I-MCP-1 binding to THP-1 cells; 31, 56, 84% decrease for 10, 50 and 100 microM resveratrol, in the absence of any effect on receptor affinity. The inhibitory effect of resveratrol on 125I-MCP-1 binding to THP-1 cells and on CCR2 protein expression determined by FACS analysis was attenuated by treatment with L-NAME (NOS inhibitor), PD98059 (MAPK inhibitor) and LY294002 (PI3K inhibitor), whereas neither X/XO (reactive oxygen species generator) nor ICI182780 (estrogen receptor antagonist) had any effect. Concomitant with a decrease in CCR2 protein expression, resveratrol inhibited THP-1 CCR2 mRNA levels, in the absence of any effect on its stability; 26 and 45% inhibition at 10 and 50 microM resveratrol, respectively. This effect was not altered by co-treatment with L-NAME, PD98059 or ICI182780, but was potentiated by LY294002 and X/XO. Conclusions: Resveratrol inhibits monocyte CCR2 binding activity in an NO-, MAPK- and PI3K-dependent manner, whereas it inhibits CCR2 mRNA in an NO- and MAPK-independent, PI3K-dependent manner. These inhibitory effects of resveratrol on chemokine receptor binding and expression may contribute, in part, to its cardiovascular protective activity in vivo.     

缺氧对人肝癌细胞株SMMC7721中HCCR表达的影响及其调控机制

背景：缺氧是实体瘤微环境的基本特征之一。人宫颈癌基因（HCCR）是新近发现的一种癌基因．在多种人类肿瘤中过表达。然而,尚缺乏HCCR在肿瘤缺氧环境中的表达及其调控机制的研究。目的：探讨缺氧对人肝癌细胞株SMMC7721中HCCR表达的影响及其调控机制。方法：SMMC7721细胞分别接受缺氧培养以及LY294002（P13K特异性抑制剂）和PD98059（MEK1特异性抑制剂）预处理＋缺氧培养,以蛋白质印迹法检测HCCR表达：将含HCCR启动子片段的荧光素酶报告基因质粒转染SMMC7721细胞,检测缺氧对HCCR启动子转录活性的影响。结果：缺氧环境下SMMC7721细胞中HCCR蛋白表达和转录活性均较常氧环境中升高,LY294002和PD98059可抑制缺氧环境下HCCR蛋白表达。结论：缺氧状态下人肝癌细胞株SMMC7721中HCCR蛋白表达和启动子转录活性增高。可能是肿瘤细胞在缺氧实体瘤组织中存活和快速增殖的重要机制之一。缺氧状态下HCCR蛋白表达受P13K／Akt和MAPK这两条信号通路的调节。     

Stimulation of insulin-like growth factor (IGF) binding protein-3 synthesis by IGF-I and transforming growth factor-alpha is mediated by both phosphatidylinositol-3 kinase and mitogen-activated protein kinase pathways in mammary epithelial cells

IGF binding protein (IGFBP)-3 is an important regulator of mammary epithelial cell (MEC) growth and can enhance the ability of both IGF-I and epidermal growth factor ligands such as TGFalpha to stimulate MEC proliferation. Here we investigate the role of the phosphatidylinositol-3 kinase (PI3K) and MAPK pathways in the regulation of IGFBP-3 expression by IGF-I and TGFalpha in bovine MECs. Both growth factors stimulated DNA synthesis, although IGF-I was the stronger mitogen. IGF-I and TGFalpha also stimulated IGFBP-3 mRNA and protein levels. TGFalpha stimulated rapid, transient activation of Akt that was maximal at 5 min and diminished by 15 min. In contrast, IGF-I-induced Akt activation was maximal between 15 and 90 min and was sustained for 6 h. Although ERK 1/2 was maximally stimulated by TGFalpha between 5 and 15 min, IGF-I did not stimulate discernible activation of ERK 1/2. In addition, TGFalpha but not IGF-I induced rapid phosphorylation of Shc, whereas only IGF-I activated insulin receptor substrate-1. Pretreatment with the PI3K inhibitor LY294002 or knockdown of p85 with small interfering RNA inhibited IGF-I or TGFalpha-stimulated IGFBP-3 expression. Similarly, MAPK kinase-1 inhibitors PD98059 and U0126 each abolished TGFalpha-stimulated increases in IGFBP-3 mRNA levels. In contrast to TGFalpha, IGF-I retained the ability to partially increase IGFBP-3 mRNA levels in the presence of MAPK kinase-1 inhibitors, indicating that IGF-I may activate alternative substrates of the PI3K pathway that are involved in IGFBP-3 regulation. In conclusion, stimulation of IGFBP-3 mRNA levels by mitogens is regulated through both the PI3K and MAPK pathways in bovine MECs.     

IGF-I signalling in bone growth: inhibitory actions of dexamethasone and IL-1beta.

OBJECTIVE: To determine if glucocorticoids and proinflammatory cytokines inhibit bone growth through a common mechanism involving impaired IGF-I signalling. DESIGN: IGF-I (100 ng/ml), dexamethasone (dex) (10(-6)M) and IL-1beta (10 ng/ml) with inhibitors of the PI3K (LY294002) and Erk 1/2 (PD98059 and UO126) IGF-I pathways (all 10 microM) were studied using the ATDC5 chondrocyte cell line and murine fetal metatarsal cultures. RESULTS: IGF-I stimulated ATDC5 chondrocyte proliferation (322%; P < 0.001 versus control). Addition of PD or LY individually to IGF-I supplemented ATDC5 cultures partially reduced proliferation by 32% (P < 0.001), and 66% (P < 0.001), respectively. PD and LY in combination blocked all IGF-I stimulated ATDC5 proliferation. LY significantly reversed IGF-I stimulatory effects on metatarsal growth (P < 0.001), whereas PD and UO treatment had no effect. IGF-I induced ATDC5 proliferation was further decreased when Dex (24%; P < 0.01) or IL-1beta (33%; P < 0.001) were added to PD but not LY cultures. Metatarsal growth inhibition by LY was unaltered by Dex or IL-1beta addition. CONCLUSIONS: Both the PI3K and Erk 1/2 pathways contributed independently to IGF-I mediated ATDC5 proliferation. However in metatarsal cultures, the Erk 1/2 pathway was not required for IGF-I stimulated growth. Dex and IL-1beta may primarily inhibit IGF-I induced bone growth through the PI3K pathway.     

Protein kinase C (PKC)-delta/-epsilon mediate the PKC/Akt-dependent phosphorylation of extracellular signal-regulated kinases 1 and 2 in MCF-7 cells stimulated by bradykinin

In this paper the signal transduction pathways evoked by bradykinin (BK) in MCF-7 breast cancer cells were investigated. BK activation of the B(2) receptor provoked: (a) the phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2); (b) the translocation from the cytosol to the membrane of the conventional protein kinase C-alpha (PKC-alpha) and novel PKC-delta and PKC-epsilon; (c) the phosphorylation of protein kinase B (PKB/ Akt); (d) the proliferation of MCF-7 cells. The BK-induced ERK1/2 phosphorylation was completely blocked by PD98059 (an inhibitor of the mitogen-activated protein kinase kinase (MAPKK or MEK)) and by LY294002 (an inhibitor of phosphoinositide 3-kinase (PI3K)), and was reduced by GF109203X (an inhibitor of both novel and conventional PKCs); G?6976, a conventional PKCs inhibitor, did not have any effect. The BK-induced phosphorylation of PKB/Akt was blocked by LY294002 but not by PD98059. Furthermore, LY294002 inhibited the BK-provoked translocation of PKC-delta and PKC-epsilon suggesting that PI3K may be upstream to PKCs. Finally, the proliferative effects of BK were blocked by PD98059, GF109203X and LY294002. These observations demonstrate that BK acts as a proliferative agent in MCF-7 cells activating intracellular pathways involving novel PKC-delta/-epsilon, PKB/Akt and ERK1/2.     

Loss of PTEN function may account for reduced proliferation pathway sensitivity to LY294002 in human prostate and bladder cancer cells

Purpose  Inhibition of phosphoinositide 3 (PI3)-kinase pathway is attractive for cancer treatment. To examine the role of the phosphatase and tensin homolog (PTEN) in the development of resistance to the treatment. Methods  We cultured human prostate cancer cells (DU145 and PC-3 cells) and bladder cancer cells (EJ-1 and UM-UC-3 cells) with a PI3-kinase inhibitor, LY294002 for more than 6 weeks and cell proliferation was studied. Activation of Akt1 and ERK was examined by immunoblotting. We introduced the wild type PTEN in UM-UC-3 cells and their proliferation along with the signaling pathways was also examined. Results  After 6 weeks, proliferation pathway sensitivity to LY294002 was reduced in cells expressing PTEN, but not in PTEN-null cells. PD98059, a MAPK/ERK kinase inhibitor, significantly inhibited proliferation of PTEN-expressing cells, but not PTEN-null cells. Stable PTEN expression in PTEN-null UM-UC-3 cells increased serum-induced ERK activation and sensitivity to PD98059-treatment, and reduced sensitivity to LY294002 after 6 weeks of exposure. Conclusions  Loss of PTEN function may protect against resistance to PI3-kinase inhibitors through an addiction to the PI3-kinase/Akt pathway.