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Many cytokines have been proposed to regulate reproduction due to their actions on hypothalamic kisspeptin cells, the main modulators of gonadotropin-releasing hormone (GnRH) neurons. Hormones such as leptin, prolactin and growth hormone are good examples of cytokines that lead to Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway activation, consequently exerting effects in kisspeptin neurons. Different studies have investigated how specific components of the JAK/STAT signaling pathway affect the functions of kisspeptin cells, but the role of the suppressor of cytokine signaling 3 (SOCS3) in mediating cytokine actions in kisspeptin cells remains unknown. Cre-Loxp technology was used in the present study to ablate Socs3 expression in kisspeptin cells (Kiss1/Socs3-KO). Then, male and female control and Kiss1/Socs3-KO mice were evaluated for sexual maturation, energy homeostasis features, and fertility. It was found that hypothalamic Kiss1 mRNA expression is significantly downregulated in Kiss1/Socs3-KO mice. Despite reduced hypothalamic Kiss1 mRNA content, these mice did not present any sexual maturation or fertility impairments. Additionally, body weight gain, leptin sensitivity and glucose homeostasis were similar to control mice. Interestingly, Kiss1/Socs3-KO mice were partially protected against lipopolysaccharide (LPS)-induced body weight loss. Our results suggest that Socs3 ablation in kisspeptin cells partially prevents the sickness behavior induced by LPS, suggesting that kisspeptin cells can modulate energy metabolism in mice in certain situations.

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Hypothalamic kisspeptin neurons are the primary modulators of gonadotropin-releasing hormone (GnRH) neurons. It has been shown that circadian rhythms driven by the suprachiasmatic nucleus (SCN) contribute to GnRH secretion. Kisspeptin neurons are potential targets of SCN neurons due to reciprocal connections with the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeN) and the arcuate nucleus of the hypothalamus (ARH). Vasoactive intestinal peptide (VIP), a notable SCN neurotransmitter, modulates GnRH secretion depending on serum estradiol levels, aging or time of the day. Considering that kisspeptin neurons may act as interneurons and mediate VIP's effects on the reproductive axis, we investigated the effects of VIP on hypothalamic kisspeptin neurons in female mice during estrogen negative feedback. Our findings indicate that VIP induces a TTX-independent depolarization of approximately 30% of AVPV/PeN kisspeptin neurons in gonad-intact (diestrus) and ovariectomized (OVX) mice. In the ARH, the percentage of kisspeptin neurons that were depolarized by VIP was even higher (approximately 90%). An intracerebroventricular infusion of VIP leds to an increased percentage of kisspeptin neurons expressing the phosphoSer133 cAMP-response-element-binding protein (pCREB) in the AVPV/PeN. On the other hand, pCREB expression in ARH kisspeptin neurons was similar between saline- and VIP-injected mice. Thus, VIP can recruit different signaling pathways to modulate AVPV/PeN or ARH kisspeptin neurons, resulting in distinct cellular responses. The expression of VIP receptors (VPACR) was upregulated in the AVPV/PeN, but not in the ARH, of OVX mice compared to mice on diestrus and estradiol-primed OVX mice. Our findings indicate that VIP directly influences distinct cellular aspects of the AVPV/PeN and ARH kisspeptin neurons during estrogen negative feedback, possibly to influence pulsatile LH secretion.

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Growth hormone (GH) deficiency is a common cause of late sexual maturation and fertility issues. To determine whether GH-induced effects on reproduction are associated with alterations in hypothalamic kisspeptin system, we studied the male reproduction in two distinct GH deficiency mouse models. In the first model, mice present GH deficiency secondary to arcuate nucleus of the hypothalamus (ARH) lesions induced by posnatal monosodium glutamate (MSG) injections. MSG-induced ARH lesions led to significant reductions in hypothalamic Ghrh mRNA expression and consequently growth. Hypothalamic Kiss1 mRNA expression and Kiss1-expressing cells in the ARH were disrupted in the MSG-treated mice. In contrast, kisspeptin immunoreactivity remained preserved in the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeN) of MSG-treated mice. Importantly, ARH lesions caused late sexual maturation and infertility in male mice. In our second mouse model, we studied animals profound GH deficiency due to a loss-of-function mutation in the Ghrhr gene (Ghrhrlit/lit mice). Interestingly, although Ghrhrlit/lit mice exhibited late puberty onset, hypothalamic Kiss1 mRNA expression and hypothalamic kisspeptin fiber density were normal in Ghrhrlit/lit mice. Despite presenting dwarfism, the majority of Ghrhrlit/lit male mice were fertile. These findings suggest that spontaneous GH deficiency during development does not compromise the kisspeptin system. Furthermore, ARH Kiss1-expressing neurons are required for fertility, while AVPV/PeN kisspeptin expression is sufficient to allow maturation of the hypothalamic-pituitary-gonadal axis in male mice.

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BACKGROUND AND AIMS: Expansion and morphological dysregulation of the bronchial vascular network occurs in asthmatic airways. Interleukin (IL) -17 and Rho-kinase (ROCK) are known to act in inflammation control and remodeling. Modulation of Rho-kinase proteins and IL-17 may be a promising approach for the treatment of asthma through the control of angiogenesis. Our objective was to analyze the effects of treatment with anti-IL17 and/or Rho-kinase inhibitor on vascular changes in mice with chronic allergic pulmonary inflammation. METHODS: Sixty-four BALB/c mice, with pulmonary inflammation induced by ovalbumin were treated with anti-IL17A (7.5/µg per dose, intraperitoneal) and/or Rho-kinase inhibitor (Y-27632-10 mg/kg, intranasal), 1 h before each ovalbumin challenge (22, 24, 26, and 28/days). Control animals were made to inhale saline. At the end of the protocol, lungs were removed, and morphometric analysis was performed to quantify vascular inflammatory, remodeling, and oxidative stress responses. RESULTS: Anti-IL17 or Rho-kinase inhibitor reduced the number of CD4+, CD8+, dendritic cells, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, Rho-kinase 1 and 2, transforming growth factor (TGF-ß), vascular endothelial growth factor (VEGF), nuclear factor (NF)-KappaB, iNOS, metalloproteinase (MMP)-9, MMP-12, metalloproteinase inhibitor-1 (TIMP-1), FOXP-3, signal transducer and activator of transcription 1 (STAT1) and phospho-STAT1-positive cells, and actin, endothelin-1, isoprostane, biglycan, decorin, fibronectin and the collagen fibers volume fraction compared with the ovalbumin group (p < 0.05). The combination treatment, when compared with anti-IL17, resulted in potentiation of decrease in the number of IL1ß- and dendritic cells-positive cells. When we compared the OVA-RHO inhibitor-anti-IL17 with OVA-RHO inhibitor we found a reduction in the number of CD8+ and IL-17, TGF-ß, and phospho-STAT1-positive cells and endothelin-1 in the vessels (p < 0.05). There was an attenuation in the number of ROCK 2-positive cells in the group with the combined treatment when compared with anti-IL17 or Rho-kinase inhibitor-treated groups (p < 0.05). CONCLUSION: We observed no difference in angiogenesis after treatment with Rho-kinase inhibitor and anti-IL17. Although the treatments did not show differences in angiogenesis, they showed differences in the markers involved in the angiogenesis process contributing to inflammation control and vascular remodeling.The reviews of this paper are available via the supplemental material section.

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Growth hormone (GH) is a key factor in the regulation of body growth, as well as a variety of other cellular and metabolic processes. Neurons expressing kisspeptin and leptin receptors (LepR) have been shown to modulate the hypothalamic-pituitary-gonadal (HPG) axis and are considered GH-responsive. The presence of functional GH receptors (GHR) in these neural populations suggests that GH may regulate the HPG axis via a central mechanism. However, there have been no studies evaluating whether or not GH-induced intracellular signaling in the brain plays a role in the timing of puberty or mediates the ovulatory cycle. Towards the goal of understanding the influence of GH on the central nervous system as a mediator of reproductive functions, GHR ablation was induced in kisspeptin and LepR expressing cells or in the entire brain. The results demonstrated that GH signaling in specific neural populations can potentially modulate the hypothalamic expression of genes related to the reproductive system or indirectly contribute to the progression of puberty. GH action in kisspeptin cells or in the entire brain was not required for sexual maturation. On the other hand, GHR ablation in LepR cells delayed puberty progression, reduced serum leptin levels, decreased body weight gain and compromised the ovulatory cycle in some individuals, while the lack of GH effects in the entire brain prompted shorter estrous cycles. These findings suggest that GH can modulate brain components of the HPG axis, although central GH signaling is not required for the timing of puberty.

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ABSTRACT Objective: To verify the physiological action of triiodothyronine T3 on the expression of transforming growth factor α (TGFA) mRNA in MCF7 cells by inhibition of RNA Polymerase II and the MAPK/ERK pathway Materials and methods: The cell line was treated with T3 at a physiological dose (10−9M) for 10 minutes, 1 and 4 hour (h) in the presence or absence of the inhibitors, α-amanitin (RNA polymerase II inhibitor) and PD98059 (MAPK/ERK pathway inhibitor). TGFA mRNA expression was analyzed by RT-PCR. For data analysis, we used ANOVA, complemented with the Tukey test and Student t-test, with a minimum significance of 5%. Results: T3 increases the expression of TGFA mRNA in MCF7 cells in 4 h of treatment. Inhibition of RNA polymerase II modulates the effect of T3 treatment on the expression of TGFA in MCF7 cells. Activation of the MAPK/ERK pathway is not required for T3 to affect the expression of TGFA mRNA. Conclusion: Treatment with a physiological concentration of T3 after RNA polymerase II inhibition altered the expression of TGFA. Inhibition of the MAPK/ERK pathway after T3 treatment does not interfere with the TGFA gene expression in a breast adenocarcinoma cell line.

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OBJECTIVE: To verify the physiological action of triiodothyronine T3 on the expression of transforming growth factor α (TGFA) mRNA in MCF7 cells by inhibition of RNA Polymerase II and the MAPK/ERK pathway. MATERIALS AND METHODS: The cell line was treated with T3 at a physiological dose (10-9M) for 10 minutes, 1 and 4 hour (h) in the presence or absence of the inhibitors, α-amanitin (RNA polymerase II inhibitor) and PD98059 (MAPK/ERK pathway inhibitor). TGFA mRNA expression was analyzed by RT-PCR. For data analysis, we used ANOVA, complemented with the Tukey test and Student t-test, with a minimum significance of 5%. RESULTS: T3 increases the expression of TGFA mRNA in MCF7 cells in 4 h of treatment. Inhibition of RNA polymerase II modulates the effect of T3 treatment on the expression of TGFA in MCF7 cells. Activation of the MAPK/ERK pathway is not required for T3 to affect the expression of TGFA mRNA. CONCLUSION: Treatment with a physiological concentration of T3 after RNA polymerase II inhibition altered the expression of TGFA. Inhibition of the MAPK/ERK pathway after T3 treatment does not interfere with the TGFA gene expression in a breast adenocarcinoma cell line.

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Background: Interleukin-17 (IL-17) and Rho-kinase (ROCK) play an important role in regulating the expression of inflammatory mediators, immune cell recruitment, hyper-responsiveness, tissue remodeling, and oxidative stress. Modulation of IL-17 and ROCK proteins may represent a promising approach for the treatment of this disease. Objective: To study the effects of an anti-IL17 neutralizing antibody and ROCK inhibitor treatments, separately and in combination, in a murine model of chronic allergy-induced lung inflammation. Methods: Sixty-four BALBc mice, were divided into eight groups (n = 8): SAL (saline-instilled); OVA (exposed-ovalbumin); SAL-RHOi (saline and ROCK inhibitor), OVA-RHOi (exposed-ovalbumin and ROCK inhibitor); SAL-anti-IL17 (saline and anti-IL17); OVA-anti-IL17 (exposed-ovalbumin and anti-IL17); SAL-RHOi-anti-IL17 (saline, ROCK inhibitor and anti-IL17); and OVA-RHOi-anti-IL17 (exposed-ovalbumin, anti-IL17, and ROCK inhibitor). A 28-day protocol of albumin treatment was used for sensitization and induction of pulmonary inflammation. The anti-IL17A neutralizing antibody (7.5 µg per treatment) was administered by intraperitoneal injection and ROCK inhibitor (Y-27632) intranasally (10 mg/kg), 1 h prior to each ovalbumin challenge (days 22, 24, 26, and 28). Results: Treatment with the anti-IL17 neutralizing antibody and ROCK inhibitor attenuated the percentage of maximal increase of respiratory system resistance and respiratory system elastance after challenge with methacholine and the inflammatory response markers evaluated (CD4+, CD8+, ROCK1, ROCK2, IL-4, IL-5, IL-6, IL-10 IL-13, IL-17, TNF-α, TGF-ß, NF-κB, dendritic cells, iNOS, MMP-9, MMP-12, TIMP-1, FOXP3, isoprostane, biglycan, decorin, fibronectin, collagen fibers content and gene expression of IL-17, VAChT, and arginase) compared to the OVA group (p < 0.05). Treatment with anti-IL17 and the ROCK inhibitor together resulted in potentiation in decreasing the percentage of resistance increase after challenge with methacholine, decreased the number of IL-5 positive cells in the airway, and reduced, IL-5, TGF-ß, FOXP3, ROCK1 and ROCK2 positive cells in the alveolar septa compared to the OVA-RHOi and OVA-anti-IL17 groups (p < 0.05). Conclusion: Anti-IL17 treatment alone or in conjunction with the ROCK inhibitor, modulates airway responsiveness, inflammation, tissue remodeling, and oxidative stress in mice with chronic allergic lung inflammation.

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Previous studies have shown that kisspeptin neurons are important mediators of prolactin's effects on reproduction. However, the cellular mechanisms recruited by prolactin to affect kisspeptin neurons remain unknown. Using whole-cell patch-clamp recordings of brain slices from kisspeptin reporter mice, we observed that 20% of kisspeptin neurons in the anteroventral periventricular nucleus was indirectly depolarized by prolactin via an unknown population of prolactin responsive neurons. This effect required the phosphatidylinositol 3-kinase signaling pathway. No effects on the activity of arcuate kisspeptin neurons were observed, despite a high percentage (70%) of arcuate neurons expressing prolactin-induced STAT5 phosphorylation. To determine whether STAT5 expression in kisspeptin cells regulates reproduction, mice carrying Stat5a/b inactivation specifically in kisspeptin cells were generated. These mutants exhibited an early onset of estrous cyclicity, indicating that STAT5 transcription factors exert an inhibitory effect on the timing of puberty.

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