The Mammalian Target of Rapamycin-p70 Ribosomal S6 Kinase but Not Phosphatidylinositol 3-Kinase-Akt Signaling Is Responsible for Fibroblast Growth Factor-9-induced Cell Proliferation

Abstract

Fibroblast growth factor-9 (FGF9) is a potent mitogen that stimulates normal and cancer cell proliferation though the signaling mechanism is not fully understood. In this study, we aimed to unravel the signaling cascades mediate FGF9 actions in human uterine endometrial stromal cell. Our results demonstrate that the mitogenic effect of FGF9 is transduced via two parallel but additive signaling pathways involving mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase. Activation of mTOR by FGF9 induces p70 ribosomal S6 kinase (S6K1) phosphorylation, cyclin expression, and cell proliferation, which are independent of phosphatidylinositol 3-kinase and Akt. Coimmunoprecipitation analysis demonstrates that mTOR physically associates with S6K1 upon FGF9 treatment, whereas ablation of mTOR activity using RNA interference or pharmacological inhibitor blocks S6K1 phosphorylation and cell proliferation induced by FGF9. Further study demonstrates that activation of mTOR is regulated by a phospholipase C␥-controlled calcium signaling pathway. These studies provide evidence to demonstrate, for the first time, that a novel signaling cascade involving phospholipase C␥, calcium, mTOR, and S6K1 is activated by FGF9 in a receptor-specific manner.Fibroblast growth factor-9 (FGF9), 1 a potent mitogen and survival factor for numerous cell types (1-6), belongs to a heparin-binding polypeptide family that consists of at least 23 structurally related proteins (7,8). Expression of FGF9 plays critical roles in sex determination, bone formation, neuron development, lens fiber differentiation, and gap junction formation (4, 9 -12). Null-allele mice lacking FGF9 exhibit severe lung hypoplasia and die shortly after birth (4). On the other hand, aberrant expression of FGF9 is associated with the development of several human diseases including prostate cancer, brain tumor, and endometriosis (3, 6, 13). Despite its well studied biological functions, the signaling mechanism of FGF9 remains to be identified.The signaling of FGF is mediated via complex interactions between specific members of the FGF family and one or more FGF receptor isoforms. Receptors for FGF (FGFR1 to -4) are tyrosine kinase receptors consisting of two intracellular tyrosine kinase domains, a single transmembrane domain, and an extracellular portion that contains three immunoglobulin-like (Ig) domains. The third Ig domain (Ig III), which exerts the highest impact on FGF receptor binding specificity and tissuespecific expression patterns, is the region in which alternative splicing occurs. Three different splice variants (designated as IIIa, IIIb, and IIIc) have been identified for FGFR1 and FGFR2, whereas only the IIIb and IIIc variants have been detected for FGFR3 (14 -17). As of yet, no splice variant for FGFR4 has been identified. The splicing variant "IIIa" of FGFR is a secreted protein, whereas IIIb and IIIc are both membrane-bound receptors containing mutually exclusive Ig III domains. It is generally believed that the IIIb isoform of FGFRs is ex...

Novel pathway in Bcr-Abl signal transduction involves Akt-independent, PLC-γ1-driven activation of mTOR/p70S6-kinase pathway

“…For instance, FGF-9 could induce a PLC-g1-controlled mTOR and p70S6-kinase activation, although PI3K and Akt were not at all activated in the analyzed human endometrial stroma cells (Wing et al, 2005). Two other examples of Aktindependent mTOR/p70S6-kinase activation are provided from transformed B lymphocytes (Wlodarski et al, 2005) and from prostaglandin-F2a-treated bovine luteal cells (Arvisais et al, 2006), even though the mechanisms involved here might be different.…”

Thyroid-Stimulating Hormone–Initiated Proliferative Signals Converge In vivo on the mTOR Kinase without Activating AKT

“…For example, it has been recently shown that prostaglandin F 2a activates mTOR via MAPK, but not via AKT in luteal cells (18), whereas mTOR activation, independent of both AKT and MAPK, has been detected in B lymphocytes (19) and endometrial stromal cells (20). The dissection of the specific mechanisms involved in the TSH-dependent activation of mTOR is tightly linked to the identification of the key players downstream of TSHR, and those upstream of mTOR.…”

Integrin-Linked Kinase Expression Is Elevated in Human Cardiac Hypertrophy and Induces Hypertrophy in Transgenic Mice

“…38 Activation of p70S6K can occur by dual activation of an mTOR-dependent pathway targeting Thr389 and an ERK1/2-dependent pathway targeting Thr421/Ser424, both of which are independent of the Akt/PKB pathway. 32,33 Further evidence in support of ILK-dependent activation of p70S6K was the finding in isolated cardiomyocytes of increased ILK-specific phosphorylation of S6 ribosomal protein, which is a downstream target of p70S6K. Our results extend the recent finding that the tripartite complex of ILK and the cytoskeletal interacting proteins particularly interesting new cysteine-histidine-rich protein and ␣-parvin is necessary for phenylalanine-induced hypertrophy in neonatal rat cardiomyocytes 39 and are consistent with the physiological hypertrophic response and augmented cardiac function associated with genetic stimulation of the ERK1/2 branch of the mitogen-activated protein kinase signaling pathway.…”

“…Adenoviral overexpression of wild-type ILK, but not the ILK R211A mutant construct or GFP control, caused activation of p70S6K at Ser371 and Thr389, which was prevented by the small-molecule ILK inhibitor KP307-2 26 ( Figure 6C). Because Thr421 and Ser424 are targets of ERK1/2, 33 we tested whether phosphorylation at these sites was ILK dependent. In concordance with the findings in the Tg mouse hearts (supplementary Figure IIIc and IIId) and human hypertrophic hearts ( Figure 3C), our results indicate that acute ILK upregulation in human fetal cardiomyocytes in vitro also induces KP307-2-inhibitable phosphorylation of p70S6K; specifically, ILK activation resulted in an increased intensity of phospho-Thr421/Ser424 and phospho-Thr389 of p70S6K, indicating cooperative regulation by ERK1/2 and mTOR kinases, respectively ( Figure 6C).…”