RESUMO
The development of proliferative podocytopathies has been linked to ligation of tumor necrosis factor receptor 2 (TNFR2) expressed on the renal parenchyma; however, the TNFR2-positive cells within the kidney responsible for podocyte injury are unknown. We detected de novo expression of TNFR2 on podocytes before hyperplastic injury in crescentic glomerulonephritis of mice with nephrotoxic nephritis, and in collapsing glomerulopathy of Tg26(HIV/nl) mice, kd/kd mice, and human beings. We further found that serum levels of soluble TNF-α and TNFR2 correlated significantly with renal injury in Tg26(HIV/nl) mice. Thus, we asked whether ligand binding of TNFR2 on podocytes ex vivo precipitates the characteristic proliferative and pro-inflammatory diseased podocyte phenotypes. Soluble TNF-α activated NF-κB and dose-dependently induced podocyte proliferation, marked by the expression of the podocyte G(1) cyclin and NF-κB target gene, cyclin D1. Microarray gene and chemokine protein expression profiling showed a marked pro-inflammatory NF-κB signature, and activated podocytes secreting CCL2- and CCL5-induced macrophage migration in transwell assays. Neutralization of TNFR2 on podocytes with blocking antibodies abrogated NF-κB activation and the induction of cyclin D1 by TNF-α, and identified TNFR2 as the primary receptor that induced IκBα degradation, the initiating event in NF-κB activation. These results suggest that TNFR2 expressed on podocytes and its canonical NF-κB signaling may directly interpose the compound pathogenic responses by podocytes to TNF-α, in the absence of other TNFR2-positive renal cell types in proliferative podocytopathies.
Assuntos
Proliferação de Células , NF-kappa B/metabolismo , Nefrite/metabolismo , Podócitos/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Perfilação da Expressão Gênica , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Rim/metabolismo , Rim/patologia , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibidor de NF-kappaB alfa , Nefrite/sangue , Nefrite/patologia , Nefrite/virologia , Análise de Sequência com Séries de Oligonucleotídeos , Podócitos/patologia , RNA Mensageiro/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/antagonistas & inibidores , Receptores Tipo II do Fator de Necrose Tumoral/sangue , Organismos Livres de Patógenos Específicos , Fator de Necrose Tumoral alfa/sangueRESUMO
Multiple lines of evidence suggest a functional link between the androgen receptor (AR) and the serine/threonine kinase Akt in the development and progression of prostate cancer. To investigate the impact of Akt activity on AR homeostasis, we treated androgen-dependent LNCaP and LAPC-4 prostate cancer cells with Akt inhibitor. Akt inhibition decreased AR expression, suggesting that Akt activity was required for regulation of AR protein levels. However, while androgen-independent LNCaP-abl cells also showed diminished AR protein levels in response to Akt inhibition, treatment of androgen-independent LNCaP-AI cells failed to alter AR protein levels upon similar treatment, suggesting that AR protein levels in these androgen-independent prostate cells were regulated by mechanisms independent of Akt activation. Regulation of AR, downstream of activated Akt, also was observed in vivo when examining transgenic mice that overexpress constitutively active mutant myristoylated (myr)-Akt1 in the prostate. Transgenic mice expressing activated myr-Akt1 exhibited higher levels of AR mRNA and protein. Expression of activated myr-Akt1 did not alter prostate cell growth and no significant size differences between prostate tissues derived from transgenic animals were observed when comparing transgenic mice with wild-type mice. Still, transgenic mice overexpressing Akt exhibited higher levels of γH2AX and phosphorylated Chk2 in prostate tissue. These changes in markers associated with oncogene-induced senescence confirmed significant altered signaling in the transgenic mouse model. Overall, results presented here suggest that AR levels are regulated by the Akt pathway.
Assuntos
Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Androgênicos/biossíntese , Androgênios/farmacologia , Animais , Western Blotting , Linhagem Celular Tumoral , Quinase do Ponto de Checagem 2 , Cromonas/farmacologia , Histonas/biossíntese , Humanos , Masculino , Metribolona/farmacologia , Camundongos , Camundongos Transgênicos , Morfolinas/farmacologia , Próstata/anatomia & histologia , Próstata/efeitos dos fármacos , Próstata/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Nuclear factor-kappaB (NF-kappaB) is a key transcriptional regulator of inflammatory bowel disease (IBD). AIM: To investigate the therapeutic potential of a locally administered "non-viral" nuclear factor-kappaB decoy (NFkappaBD) in multiple experimental models of IBD. METHODS: A fully phosphorothioated decoy oligonucleotide with improved stability that specifically binds NF-kappaB and blocks inflammatory mediators regulated by this transcription factor without the help of viral envelope-assisted delivery was developed. The therapeutic effects of NFkappaBD were studied in the trinitrobenzene sulphonic acid, oxazolone and dextran sodium sulphate induced colitis models. RESULTS: Intracolonic administration of NFkappaBD results in the delivery of NFkappaBD to inflammatory cells and a reduction of NF-kappaB heterodimers. In the T helper cell 1-driven trinitrobenzene sulphonic acid-induced colitis model, mice receiving NFkappaBD treatment exhibit a dose-dependent reduction in disease severity and a more rapid recovery to normal body weight, similar to a clinically relevant dose of budesonide. Clinical efficacy was corroborated by considerable reductions in colitis pathology and tissue levels of several pro-inflammatory markers, including tumour necrosis factor alpha, interleukin 6, interleukin 1beta and monocyte chemotactic protein 1. NFkappaBD also mitigates disease activity in the T helper cell 2-like oxazolone colitis and epithelial injury-related acute dextran sodium sulphate colitis models. Interestingly, restoration of tissue homeostasis is observed in NFkappaBD-treated animals with the rapid re-emergence of functional goblet cells and a return to normal patterns of cell proliferation in the mucosal epithelium and smooth muscle cell layers. CONCLUSIONS: These data support the potential use of "naked" NFkappaBD as a cross-functional therapeutic in IBD, and show for the first time that it can facilitate the restoration of colon homeostasis and function.