脂多糖对人支气管上皮细胞STAT1、STAT3、STAT4、STAT6表达的影响

目的观察脂多糖对人支气管上皮细胞16HBESTAT1、STAT3、STAT4、STAT6表达的影响。方法采用普通RT—PCR检测16HBE细胞STAT1、STAT3、STAT4、STAT6的mRNA表达;Western印迹检测16HBE细胞STAT1、STAT4、STAT6的蛋白表达。分别采用不同浓度的脂多糖在不同的时间点处理16HBE细胞,采用Real—timePCR的方法检测16HBE细胞STAT1、STAT3、STAT4、STAT6的mRNA表达。结果1μg／m1的LPS处理16HBE细胞1h组、0．25μg／m1的LPS处理16HBE细胞4h组、1μg／ml的LPS处理16HBE细胞4h组STAT1、STAT4的mRNA表达较正常对照组显著增高（P〈0．01）;0．25μg／ml的LPS处理16HBE细胞2h组、1μg／ml的LPS处理16HBE细胞2h组、10μg／ml的LPS处理16HBE细胞2h组STAT1、STAT4的mRNA表达较正常对照组有所增高（P〈0．05）;1μg/ml的LPS处理16HBE细胞1h组STAT6的mRNA表达较正常对照组显著增高（P〈0．01）。所有LPS处理16HBE细胞组STAT3的mRNA表达均较正常对照组减低。结论人支气管上皮细胞表达STAT1、STAT3、STAT4、STAT6的mRNA和STAT1、STAT4、STAT6的蛋白,一定剂量的脂多糖在某些时间点分别刺激了人支气管上皮细胞STAT1、sTAT4、STAT6的mRNA表达。     

Cutting edge: role of STAT1, STAT3, and STAT5 in IFN-alpha beta responses in T lymphocytes

Engagement of the IFN-alphabeta receptor initiates multiple signaling cascades, including activation of the STAT. In this study, we demonstrate that IFN-alphabeta, although antiproliferative in wild-type CD4(+) or CD8(+) T cells, act as strong mitogens on their STAT1(-/-) counterparts. Furthermore, IFN-alphabeta exert little effect on apoptosis in wild-type cells, but are potent survival factors in the absence of STAT1. The antiapoptotic response in the absence of STAT1 is predominantly mediated by STAT3, and to a lesser extent by STAT5A/B. In contrast, the mitogenic IFN-alphabeta response gained through the absence of STAT1 is only marginally affected when STAT5A/B expression is also abrogated, but is completely dependent on STAT3 activation. These findings provide the first evidence for a function of STAT3 and STAT5A/B in the IFN-alphabeta response, and support a model in which the IFN-alphabeta receptor initiates both pro- and antiapoptotic responses through STAT1, and STAT3 and STAT5A/B, respectively.     

Distinct roles for STAT1, STAT3, and STAT5 in differentiation gene induction and apoptosis inhibition by interleukin-9.

Interleukin-9 (IL-9) activates three distinct STAT proteins: STAT1, STAT3, and STAT5. This process depends on one tyrosine of the IL-9 receptor, which is necessary for proliferation, gene induction, and inhibition of apoptosis induced by glucocorticoids. By introduction of point mutations in amino acids surrounding this tyrosine, we obtained receptors that activated either STAT5 alone or both STAT1 and STAT3, thus providing us with the possibility to study the respective roles of these factors in the biological activities of IL-9. Both mutant receptors were able to prevent apoptosis, but only the mutant that activated STAT1 and STAT3 was able to support induction of granzyme A and L-selectin. In line with these results, constitutively activated STAT5 blocked glucocorticoid-induced apoptosis. In Ba/F3 cells, significant proliferation and pim-1 induction were observed with both STAT-restricted mutants, though proliferation was lower than with the wild-type receptor. These results suggest that survival and cell growth are redundantly controlled by multiple STAT factors, whereas differentiation gene induction is more specifically correlated with individual STAT activation by IL-9.     

Loss of STAT1 protects hair cells from ototoxicity through modulation of STAT3, c-Jun,Akt, and autophagy factors

Hair cell damage is a side effect of cisplatin and aminoglycoside use. The inhibition or attenuation of this process is a target of many investigations. There is growing evidence that STAT1 deficiency decreases cisplatin-mediated ototoxicity; however, the role of STAT function and the molecules that act in gentamicin-mediated toxicity have not been fully elucidated. We used mice lacking STAT1 to investigate the effect of STAT1 ablation in cultured organs treated with cisplatin and gentamicin. Here we show that ablation of STAT1 decreased cisplatin toxicity and attenuated gentamicin-mediated hair cell damage. More TUNEL-positive hair cells were observed in explants of wild-type mice than that of STAT1^−/− mice. Although cisplatin increased serine phosphorylation of STAT1 in wild-type mice and diminished STAT3 expression in wild-type and STAT1^−/− mice, gentamicin increased tyrosine phosphorylation of STAT3 in STAT1^−/− mice. The early inflammatory response was manifested in the upregulation of TNF-α and IL-6 in cisplatin-treated explants of wild-type and STAT1^−/− mice. Expression of the anti-inflammatory cytokine IL-10 was altered in cisplatin-treated explants, upregulated in wild-type explants, and downregulated in STAT1^−/− explants. Cisplatin and gentamicin triggered the activation of c-Jun. Activation of Akt was observed in gentamicin-treated explants from STAT1^−/− mice. Increased levels of the autophagy proteins Beclin-1 and LC3-II were observed in STAT1^−/− explants. These data suggest that STAT1 is a central player in mediating ototoxicity. Gentamicin and cisplatin activate different downstream factors to trigger ototoxicity. Although cisplatin and gentamicin triggered inflammation and activated apoptotic factors, the absence of STAT1 allowed the cells to overcome the effects of these drugs.The process of auditory sensorineural damage implicates a variety of intracellular events caused by aging, noise exposure, aminoglycoside antibiotics, or the chemotherapeutic agent cisplatin. The mechanisms underlying the ototoxic effects of cisplatin and gentamicin are not yet completely understood. Their ototoxicity likely involves morphological changes and the modulation of pro- and anti-apoptotic cell responses.¹Activation of oxidative stress and the inflammatory response are common effects of cisplatin- and gentamicin-induced ototoxicity.² Cisplatin increased the early release of pro-inflammatory cytokines in HEI-OC1 cells and in the cochlea of cisplatin-injected rats.³ Similarly, gentamicin induced the production of pro-inflammatory cytokines in the organ of Corti explants in vitro.⁴ The JAK/STAT pathway is one of the best-characterized cellular signaling pathways in the immune system. STAT1, a regulator of cell death, has been reported to be involved in cisplatin-mediated hair cell damage.^{5, 6} Knockdown of the STAT1 gene by means of siRNA, administrated by transtympanic injection in rats and transfection of UB/OC1 cells, reduced cisplatin-induced hair cell death in vivo and in vitro. Moreover, STAT1 siRNA preserved hearing in cisplatin-treated rats.⁵ Furthermore, STAT1 phosphorylation has been observed in utricles exposed to cisplatin in vitro.⁶The inactivation of STAT1 in other tissues has also demonstrated a protective effect, for example, by enhancing autophagy in STAT1-deficient hearts⁷ or accelerated skeletal muscle regeneration.⁸ Recent findings demonstrated that inhibition of the JAK2/STAT3 signaling pathway protects against noise-induced damage to cochlear tissue⁹ and STAT3/SOCS3 signaling regulate hair cell regeneration.¹⁰ Generally, STAT1 and STAT3 are reciprocally regulated, and disruption of their balance directs cells from survival to apoptotic cell death or from inflammatory to anti-inflammatory responses.¹¹ However, there is no information about the role of STAT1 in gentamicin-induced hair cell damage.In the present study, we investigated the impact of the genetic ablation of STAT1 on hair cell damage induced by cisplatin and gentamicin. We also examined a subset of cell signaling mediators involved in apoptosis and survival. Our data indicate that STAT1 has an important role in cisplatin- and gentamicin-mediated hair cell death. We observed differences in the expression of STAT1 and STAT3 in the organ of Corti (OC) from wild-type (WT) and STAT1^−/− mice exposed to cisplatin or gentamicin. An early inflammatory response was observed in the cisplatin-treated explants. Finally, we demonstrated regulatory changes of Akt, c-Jun, and autophagy factors in OC explants exposed either to cisplatin or gentamicin.     

A preferential role for STAT5, not constitutively active STAT3, in promoting survival of a human lymphoid tumor

STATs are believed to play key roles in normal and abnormal cell function. In the present work, we investigated the role of STATs in an IL-2-responsive human lymphoblastic lymphoma-derived cell line, YT. Only STAT3 was found constitutively tyrosine phosphorylated, but not other STATs. Hyperactive STAT3 was not attributable to a pre-existing intermediate affinity IL-2R complex and/or hyperactive Jak activity. Depletion of STAT3 protein expression reduced tumor cell viability with protracted kinetics (72-96 h), while TUNEL assays demonstrated cell death occurred via apoptosis. Interestingly, depletion of STAT5 in this same tumor induced more pronounced cell death compared with STAT3 depletion (24 h). Although IL-2 was able to rescue STAT3-depleted cells from death, it could not compensate for the loss of STAT5. To determine the prosurvival function of STAT3 vs STAT5 within the same tumor model, genes were profiled in STAT3- or STAT5-depleted YT cells by apoptosis-specific microarrays. Several differentially expressed genes were identified. Interestingly, those genes involved in NF-kappaB regulation, such as TNFR-associated factors 2 and 5 and B cell leukemia/lymphoma 10, were readily decreased upon STAT5, but not STAT3, depletion as validated by quantitative RT-PCR. These results suggest that STAT5 and, to a lesser extent, hyperactive STAT3 provide preferential and critical cell survival signals for certain human lymphoid tumors, indicating that nonhyperactive STATs should be considered as therapeutic targets for abrogating tumorigenesis.     

Design, synthesis, and studies of small molecule STAT3 inhibitors

A series of small molecule STAT3 inhibitors originally derived from our lead compound STA 21 were synthesized and evaluated. The most potent compound in this series, compound 1, exhibited the same anti-proliferative activities as STA 21 against prostate cancer cell lines that express constitutively active STAT3. Molecular docking showed compound 1 bound to the STAT3beta SH2 domain in a similar manner as STA 21.