Reorganization of the actin cytoskeleton in response to growth factor signaling, such as transforming growth factor ␤ (TGF-␤), controls cell adhesion, motility, and growth of diverse cell types. In Swiss3T3 fibroblasts, a widely used model for studies of actin reorganization, TGF-␤1 induced rapid actin polymerization into stress fibers and concomitantly activated RhoA and RhoB small GTPases. Consequently, dominant-negative RhoA and RhoB mutants blocked TGF-␤1-induced actin reorganization. Because Rho GTPases are known to regulate the activity of LIM-kinases (LIMK), we found that TGF-␤1 induced LIMK2 phosphorylation with similar kinetics to Rho activation. Cofilin and LIMK2 co-precipitated and cofilin became phosphorylated in response to TGF-␤1, whereas RNA interference against LIMK2 blocked formation of new stress fibers by TGF-␤1. Because the kinase ROCK1 links Rho GTPases to LIMK2, we found that inhibiting ROCK1 activity blocked completely TGF-␤1-induced LIMK2/cofilin phosphorylation and downstream stress fiber formation. We then tested whether the canonical TGF-␤ receptor/Smad pathway mediates regulation of the above effectors and actin reorganization. Adenoviruses expressing constitutively activated TGF-␤ type I receptor led to robust actin reorganization and Rho activation, whereas the constitutively activated TGF-␤ type I receptor with mutated Smad docking sites (L45 loop) did not affect either actin organization or Rho activity. In line with this, ectopic expression of the inhibitory Smad7 inhibited TGF-␤1-induced Rho activation and cytoskeletal reorganization. Our data define a novel pathway emanating from the TGF-␤ type I receptor and leading to regulation of actin assembly, via the kinase LIMK2.

serum-and-glucocorticoid-inducible-kinase-1 (sGK1) is under regulation of several hormones, mediators and cell stressors. More specifically, sGK1 expression is particularly sensitive to glucocorticoids, mineralocorticoids, and TGFβ. Moreover, SGK1 expression is exquisitely sensitive to hypertonicity, hyperglycemia, and ischemia. sGK1 is activated by insulin and growth factors via phosphatidylinositol-3-kinase, 3-phosphoinositide dependent-kinase PDK1, and mTOr. sGK1 up-regulates the Na + /K + -ATPase, a variety of carriers (e.g. Ncc, NKcc, NHE1, NHE3, sGLT1, several amino acid transporters) and many ion channels (e.g. ENac, scN5A, TrPV4-6, Orai1/sTIM1, rOMK, KcNE1/KcNQ1, Glur6, cFTr). sGK1 further up-regulates a number of enzymes (e.g. glycogen-synthase-kinase-3, ubiquitin-ligase Nedd4-2), and transcription factors (e.g. forkhead-transcription-factor FOXO3a, β-catenin, nuclear-factor-kappa-B NFκB). SGK1 sensitive functions contribute to regulation of epithelial transport, excitability, degranulation, matrix protein deposition, coagulation, platelet aggregation, migration, cell proliferation, and apoptosis. Apparently, SGK1 is not required for housekeeping functions, as the phenotype of sGK1 knockout mice is mild. However, excessive sGK1 expression and activity participates in the pathophysiology of several disorders, including hypertension, obesity, diabetes, thrombosis, stroke, inflammation, autoimmune disease, fibrosis, and tumor growth. A sGK1 gene variant (prevalence ~3-5% prevalence in caucasians, ~10% in Africans) predisposes to hypertension, stroke, obesity, and type 2 diabetes. Moreover, excessive salt intake and/or excessive release of glucocorticoids, mineralocorticoids, and TGFβ up-regulates SGK1 expression thus predisposing to sGK1-related diseases.

Recent findings have shown that, in addition to the genomic action of steroids, through intracellular receptors, short-time effects could be mediated through binding to membrane sites. In the present study of prostate cancer LNCaP cells, we report that dihydrotestosterone and the non-internalizable analog testosterone-BSA increase rapidly the release of prostate-specific antigen (PSA) in the culture medium. Membrane testosterone binding sites were identified through ligand binding on membrane preparations, flow cytometry, and confocal laser microscopy of the non-internalizable fluorescent analog testosterone-BSA-FITC, on whole cells. Binding on these sites is time- and concentration-dependent and specific for testosterone, presenting a KD of 10.9 nM and a number of 144 sites/mg protein (approximately 13000 sites/cell). Membrane sites differ immunologically for intracellular androgen receptors. The secretion of PSA after membrane testosterone receptor stimulation was inhibited after pretreatment with the actin cytoskeleton disrupting agent cytochalasin B. In addition, membrane testosterone binding modifies the intracellular dynamic equilibrium of monomeric to filamentous actin and remodels profoundly the actin cytoskeleton organization. These results are discussed in the context of a possible involvement of these sites in cancer chemotherapy.

The human prostate cancer cell line LNCaP bears functional membrane testosterone receptors, which modify the actin cytoskeleton and increase the secretion of prostate-specific antigen (PSA) within minutes. Membrane steroid receptors are, indeed, a newly identified element of steroid action that is different from the classical intracellular sites. In the present work, using a nonpermeable analog of testosterone (testosterone-BSA), we investigated the signaling pathway that is triggered by the membrane testosterone receptors' activation and leads to actin cytoskeleton reorganization. We report that exposure of cells to testosterone-BSA resulted in phosphorylation of focal adhesion kinase (FAK), the association of FAK with the phosphatidylinositol-3 (PI-3) kinase, and the subsequent activation of the latter as well as the activation of the small guanosine triphosphatases Cdc42/Rac1. Pretreatment of cells with the specific PI-3 kinase inhibitor wortmannin abolished both the activation of the small guanosine triphosphatases and the alterations of actin cytoskeleton, whereas it did not affect the phosphorylation of FAK. These findings indicate that PI-3 kinase is activated downstream of FAK and upstream of Cdc42/Rac1, which subsequently regulate the actin organization. Moreover, wortmannin diminished the secretion of PSA, implying that the signaling events described above are responsible for the testosterone-BSA-induced PSA secretion. Our results are discussed under the prism of a possible implication of these membrane receptors in prostate cancer chemotherapy.

Nongenomic androgen actions imply mechanisms different from the classical intracellular androgen receptor (iAR) activation. We have recently reported the identification of a membrane androgen receptor (mAR) on LNCaP human prostate cancer cells, mediating testosterone signal transduction within minutes. In the present study we provide evidence that activation of mAR by nonpermeable, BSA-coupled testosterone results in 1) inhibition of LNCaP cell growth (with a 50% inhibitory concentration of 5.08 nM, similar to the affinity of testosterone for membrane sites); 2) induction in LNCaP cells of both apoptosis and the proapoptotic Fas protein; and 3) a significant decrease in migration, adhesion, and invasion of iAR-negative DU145 human prostate cancer cells. These actions persisted in the presence of antiandrogen flutamide or after decreasing the content of iAR in LNCaP cells by iAR antisense oligonucleotides. Testosterone-BSA was also effective in inducing apoptosis of DU145 human prostate cancer cells, negative for iAR, but expressing mAR sites. In LNCaP cell-inoculated nude mice, treatment with testosterone-BSA (4.8 mg/kg body weight) for 1 month resulted in a 60% reduction of tumor size compared with that in control animals receiving only BSA, an effect that was not affected by the antiandrogen flutamide. Our findings suggest that activators of mAR may represent a new class of antitumoral agents of prostate cancer.

The family of p21-activated kinases (PAKs) have been implicated in the rearrangement of actin cytoskeleton by acting downstream of the small GTPases Rac and Cdc42. Here we report that even though Cdc42/Rac1 or Akt are not activated, phosphatidylinositol-3 (PI-3) kinase activation induces PAK1 kinase activity. Indeed, we demonstrate that PI-3 kinase associates with the N-terminal regulatory domain of PAK1 (amino acids 67-150) leading to PAK1 activation. The association of the PI-3 kinase with the Cdc42/Rac1 binding-deficient PAK1(H83,86L) confirms that the small GTPases are not involved in the PI-3 kinase-PAK1 interaction. Furthermore, PAK1 was activated in cells expressing the dominant-negative forms of Cdc42 or Rac1. Additionally, we show that PAK1 phosphorylates actin, resulting in the dissolution of stress fibers and redistribution of microfilaments. The phosphorylation of actin was inhibited by the kinase-dead PAK1(K299R) or the PAK1 autoinhibitory domain (PAK1(83-149)), indicating that PAK1 was responsible for actin phosphorylation. We conclude that the association of PI-3 kinase with PAK1 regulates PAK1 kinase activity through a Cdc42/Rac1-independent mechanism leading to actin phosphorylation and cytoskeletal reorganization.

We have recently identified a specific signaling pathway that regulates actin reorganization in malignant human breast and prostate epithelial cells associated with FAK, PI-3K and Rac1 activation. Here we report that this pathway operates in MCF7 cells upon activation of membrane androgen receptors (mAR). Stimulation of mAR by the non-permeable testosterone-BSA conjugate resulted in early actin reorganization documented by quantitative measurements of actin dynamics and morphological analysis of microfilament organization. This effect was regulated by early phosphorylation of FAK and subsequent PI-3K and Rac1 activation. The functional role of this pathway was further shown in A375 melanoma cells. Treatment with the opioid antagonist α_s1 casomorphin resulted in rapid and potent actin remodeling in A375 cells, regulated by rapid activation of the FAK/PI-3K/Rac1 signaling. Pretreatment of both cell lines with the specific PI-3K inhibitor wortmannin blocked actin reorganization. Interestingly, wound healing assays revealed that testosterone-BSA and α _s1 casomorphin significantly inhibited MCF7 and A375 cell motility respectively. These effects were abrogated through blockade of PI-3K signaling by wortmannin. The results presented here indicate that actin reorganization through FAK/PI3-K/Rac-1 activation operates in various human cancer cell systems supporting a functional role for FAK/PI-3K/Rac1/actin signaling in controlling cell motility.

Introduction The phosphoinositide-3 kinase (PI3K)/Akt pathway, operating downstream of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER)2, is implicated in cell migration and survival. EGFR and HER2 are expressed in circulating tumor cells, but the activation status of downstream signaling molecules has not yet been reported.