Androgens regulate protein kinase Cdelta transcription and modulate its apoptotic function in prostate cancer cells.

Activation of protein kinase Cdelta (PKCdelta), a member of the novel PKC family, leads to apoptosis in several cell types. Although the molecular bases of PKCdelta activation are being unfolded, limited information is available on the mechanisms that control its expression. Here, we report that in prostate cancer cells PKCdelta is tightly regulated by androgens at the transcriptional level. Steroid depletion from the culture medium causes a pronounced down-regulation of PKCdelta protein and mRNA in androgen-sensitive LNCaP prostate cancer cells, an effect that is rescued by the androgen R1881 in an androgen receptor (AR)-dependent manner. Analysis of the PKCdelta promoter revealed a putative androgen responsive element (ARE) located 4.7 kb upstream from the transcription start site. Luciferase reporter assays show that this element is highly responsive to androgens, and mutations in key nucleotides in the AR-binding consensus abolish reporter activity. Furthermore, using chromatin immunoprecipitation assays, we determined that the AR binds in vivo to the PKCdelta ARE in response to androgen stimulation. Functional studies revealed that, notably, androgens modulate phorbol 12-myristate 13-acetate (PMA)-induced apoptosis in LNCaP cells, an effect that is dependent on PKCdelta. Indeed, androgen depletion or AR RNA interference severely impaired the apoptotic function of PKCdelta or the activation of p38, a downstream effector of PKCdelta in LNCaP cells--effects that can be rescued by restoring PKCdelta levels using an adenoviral delivery approach. Our studies identified a novel hormonal mechanism for the control of PKCdelta expression via transcriptional regulation that fine-tunes the magnitude of PKCdelta apoptotic responses.

Protein kinase C and prostate carcinogenesis: targeting the cell cycle and apoptotic mechanisms.

A series of both genetic and epigenetic factors have been implicated in the genesis and progression of prostate cancer. Recent evidence revealed that protein kinase C (PKC) isozymes play a crucial role in the control of cell proliferation and apoptosis in prostate cancer models, as well as in the transition from an androgen-dependent to an androgen-independent status. Indeed, PKCalpha and PKCdelta promote apoptosis in androgen-dependent prostate cancer cells. Due to the relevance of PKC isozymes in the control of cell cycle, both in G1/S and G2/M, the elucidation of such complex intracellular networks using cellular and animal models has become of outmost importance. In this review, we present the current knowledge on the regulation of apoptosis and tumorigenicity by PKC isozymes and the functional roles of cell cycle regulators in prostate carcinogenesis. The development of animal models where overexpression of discrete PKCs or cell cycle regulators is targeted to the prostate will greatly contribute to the understanding of the molecular basis of the disease, and more importantly, it will have profound implications for the development of novel strategies for prostate cancer therapy.

Protein kinase C promotes apoptosis in LNCaP prostate cancer cells through activation of p38 MAPK and inhibition of the Akt survival pathway.

Activation of protein kinase C (PKC) by phorbol esters or diacylglycerol mimetics induces apoptosis in androgen-dependent prostate cancer cells, an effect that involves both the activation of the classic PKC alpha and the novel PKC delta isozymes (Fujii, T., García-Bermejo, M. L., Bernabó, J. L., Caamaño, J., Ohba, M., Kuroki, T., Li, L., Yuspa, S. H., and Kazanietz, M. G. (2000) J. Biol. Chem. 275, 7574-7582 and Garcia-Bermejo, M. L., Leskow, F. C., Fujii, T., Wang, Q., Blumberg, P. M., Ohba, M., Kuroki, T., Han, K. C., Lee, J., Marquez, V. E., and Kazanietz, M. G. (2002) J. Biol. Chem. 277, 645-655). In the present study we explored the signaling events involved in this PKC-mediated effect, using the androgen-dependent LNCaP cell line as a model. Stimulation of PKC by phorbol 12-myristate 13-acetate (PMA) leads to the activation of ERK1/2, p38 MAPK, and JNK in LNCaP cells. Here we present evidence that p38 MAPK, but not JNK, mediates PKC-induced apoptosis. Because LNCaP cells have hyperactivated Akt function due to PTEN inactivation, we examined whether this survival pathway could be affected by PKC activation. Interestingly, activation of PKC leads to a rapid and reversible dephosphorylation of Akt, an effect that was prevented by the pan-PKC inhibitor GF109302X and the cPKC inhibitor Gö6976. In addition, the diacylglycerol mimetic agent HK654, which selectively stimulates PKC alpha in LNCaP cells, also induced the dephosphorylation of Akt in LNCaP cells. Inactivation of Akt function by PKC does not involve the inhibition of PI3K, and it is prevented by okadaic acid, suggesting the involvement of a phosphatase 2A in PMA-induced Akt dephosphorylation. Finally, we show that, when an activated form of Akt is delivered into LNCaP cells by either transient transfection or adenoviral infection, the apoptotic effect of PMA is significantly reduced. Our results highlight a complex array of signaling pathways regulated by PKC isozymes in LNCaP prostate cancer cells and suggest that both p38 MAPK and Akt play critical roles as downstream effectors of PKC isozymes in this cellular model.

Na(+)/I(-) symporter activity requires a small and uncharged amino acid residue at position 395.

Active iodide uptake in the thyroid is mediated by the Na(+)/I(-) symporter (NIS), a key plasma membrane glycoprotein. Several NIS mutations have been shown to cause I(-) transport defect, a condition that, if untreated, can lead to congenital hypothyroidism and, ultimately, cretinism. The study of I(-) transport defect-causing NIS mutations provides valuable insights into the structure-function and mechanistic properties of NIS. Here we report the thorough analysis of the G395R NIS mutation. We observed no I(-) uptake activity at saturating or even supersaturating external I(-) concentrations in COS-7 cells transiently transfected with G395R NIS cDNA, even though we demonstrated normal expression of G395R NIS and proper targeting to the plasma membrane. Several amino acid substitutions at position 395 showed that the presence of an uncharged amino acid residue with a small side chain at position 395 is required for NIS function, suggesting that glycine 395 is located in a tightly packed region of NIS. Substitutions of large amino acid residues at position 395 resulted in lower V(max) without affecting K(m) values for I(-) and Na(+), suggesting that these residues hamper the Na(+)/I(-) coupling reaction.