Ebru Karpuzoglu

Estrogen regulates transcription factors STAT-1 and NF-kappaB to promote inducible nitric oxide synthase and inflammatory responses.

Estrogen regulation of inflammatory responses has broad physiological and pathological consequences. However, the molecular mechanism of estrogen regulation of inflammation is still poorly understood. In this study, we report that activation of both STAT-1 and NF-κB signaling is essential for Con A-induced inducible NO synthase (iNOS) and NO in murine splenocytes. Estrogen enhances STAT-1 DNA-binding activity without increasing the expression of phosphorylated and total STAT-1 protein. We have recently reported that estrogen blocks the nuclear expression of NF-κB p65 and modifies nuclear NF-κBp50. Here, we demonstrated that both nuclear STAT-1 and NF-κB are modified by serine protease-mediated proteolysis, which resulted in altered STAT-1 and NF-κB activity/signaling in splenocytes from estrogen-treated mice. Inhibition of serine protease activity with 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) restores the nuclear expression of full-length STAT-1 and NF-κB proteins, and resulted in decreased STAT-1 DNA-binding activity and formation of NF-κB p65/p50 binding complexes in nuclei of splenocytes from estrogen-treated mice. Consequently, there is significantly decreased iNOS and IFN-γ production in AEBSF-treated splenocytes from estrogen-treated mice, which suggests a positive regulatory role of truncated STAT-1 and/or NF-κB. Interestingly, there is increased production of MCP-1 in STAT-1 or NF-κB small interfering RNA-transfected cells, as well as in AEBSF-treated splenocytes from estrogen-treated mice. These data suggest a differential role of truncated STAT-1 and NF-κB in regulation of various inflammatory molecules in splenocytes from estrogen-treated mice. Together, our data reveal a novel molecular mechanism of estrogen-mediated promotion of inflammatory responses, which involves posttranslational modification of STAT-1 and NF-κB proteins.

The Multi-faceted Influences of Estrogen on Lymphocytes: Toward Novel Immuno-interventions Strategies for Autoimmunity Management

Early studies of the immune system disclosed that, generally, females exhibit stronger responses to a variety of antigens than males. Perhaps as a result of this response, women are more prone to developing autoimmune diseases than men. Yet, the precise cellular and molecular mechanisms remain under investigation. Recently, interferon-gamma and the related pro-inflammatory interleukin-12 were found to be under effects of sex steroid hormones, with potential implications in regulating immune cells and autoimmune responses. In B lymphocytes, functional binding sites for estrogen receptors were identified in the promoter of the gene encoding activation-induced deaminase, an enzyme required for somatic hypermutation, and class-switch recombination. The observation that estrogen exerts direct impacts on antibody affinity-maturation provides a potential mechanism that could account for generating pathogenic high-affinity auto-antibodies. Further deciphering the multi-faceted influences of sex hormones on the responsiveness of immune cells could lead to novel therapeutic interventions for autoimmunity management.

Estrogen selectively regulates chemokines in murine splenocytes

Estrogen has striking effects on immunity and inflammatory autoimmune conditions. One potential mechanism of estrogen‐induced regulation of immunity and inflammatory autoimmune conditions is by altering the secretion of chemokines by lymphocytes, an aspect not well addressed thus far. We found that estrogen has marked, but differential, effects on the secretion of chemokines from activated splenocytes. Estrogen treatment significantly increased the secretion of MCP‐1, MCP‐5, eotaxin, and stromal cell‐derived factor 1β from Con A‐activated splenocytes when compared with placebo‐treated controls, and it had no effects on the levels of RANTES, thymus and activation‐regulated chemokine, and keratinocyte‐derived chemokine (KC) at 24 h. A kinetic analysis showed that chemokines tended to increase with stimulation time, but only MCP‐1 and MCP‐5 showed a biological trend of increasing in splenocytes from estrogen‐treated mice, and KC was decreased significantly in estrogen‐treated splenocytes at 18 h. Estrogen did not affect the protein levels of chemokine receptors CCR1 or CCR2 at 24 h. Estrogen‐induced alterations in the levels of MCP‐1 and MCP‐5 are mediated, in part, by IFN‐γ, as estrogen treatment of IFN‐γ null mice, unlike wild‐type mice, did not up‐regulate these chemokines. However, addition of recombinant IFN‐γ resulted in markedly increased secretion of MCP‐1 and MCP‐5 only in the cells derived from estrogen‐treated mice. These studies provide novel data indicating that estrogen may promote inflammatory conditions by altering the levels of chemokines, providing evidence for an additional mechanism by which estrogens can regulate inflammation.

Effects of Sex Steroids on Innate and Adaptive Immunity

Estrogens and androgens are classically recognized as reproductive sex steroid hormones because of their well-documented effects on reproductive tissues. However, extensive research in diverse biological disciplines have clearly established that reproductive hormones have broad physiological effects on nonreproductive tissues, including the immune, central nervous, cardiovascular, and skeletal systems. Thus, the term “sex/reproductive hormones” describes only a narrow (albeit important) aspect of biological effects of sex steroids. In this chapter, the effects of sex hormones on the innate and adaptive immune system are highlighted. Generally, estrogens upregulate proinflammatory cytokines (e.g., IFNγ) and IFNγ-inducible molecules (nitric oxide, NOS2, and COX2), whereas androgens suppress proinflammatory responses. Immunomodulation by sex steroids may have both physiological and pathological implications (e.g., sex differences in immune capabilities and in inflammatory diseases, respectively).

Signal Transducer and Activation of Transcription (STAT) 4β, a Shorter Isoform of Interleukin-12-Induced STAT4, Is Preferentially Activated by Estrogen

Estrogen, a natural immunomodulatory compound, has been shown to promote the induction of a prototype T helper 1 cytokine, interferon (IFN)-gamma, as well as to up-regulate IFNgamma-mediated proinflammatory molecules (nitric oxide, cyclooxygenase 2, monocyte chemoattractant protein 1). Because IL-12 is a major IFNgamma-inducing cytokine, in this study we investigated whether estrogen treatment of wild-type C57BL/6 mice alters IL-12-mediated signaling pathways. A recent study has shown that IL-12 activates two isoforms of signal transducer and activation of transcription (STAT) 4, a normal-sized (full-length STAT4alpha) and a truncated form (STAT4beta). Interestingly, we found that estrogen treatment preferentially up-regulates the phosphorylation of STAT4beta in splenic lymphoid cells. Time kinetic data showed the differential activation of STAT4beta in splenic lymphoid cells from estrogen-treated mice, but not in cells from placebo controls. The activation of STAT4beta was mediated by IL-12 and not IFNgamma because deliberate addition or neutralization of IL-12, but not IFNgamma, affected the activation of STAT4beta. In contrast to IL-12-induced activation of STAT4beta in cells from estrogen-treated mice, STAT4alpha was not increased, rather it tended to be decreased. In this context, STAT4alpha-induced p27(kip1) protein was decreased in concanavalin A + IL-12-activated lymphocytes from estrogen-treated mice only. By using the in vitro DNA binding assay, we confirmed the ability of pSTAT4beta to bind to the IFNgamma-activated sites (IFNgamma activation sequences)/STAT4-binding sites in estrogen-treated mice. Our data are the first to show that estrogen apparently has selective effects on IL-12-mediated signaling by preferentially activating STAT4beta. These novel findings are likely to provide new knowledge with regard to estrogen regulation of inflammation.