No abstract

Melatonin is a key hormone that regulates circadian rhythms, metabolism, and reproduction. However, the mechanisms of melatonin synthesis and secretion have not been fully defined. The purpose of this study was to investigate the functions of the LIM homeobox transcription factor Isl1 in regulating melatonin synthesis and secretion in porcine pineal gland. We found that Isl1 is highly expressed in the melatonin-producing cells in the porcine pineal gland. Further functional studies demonstrate that Isl1 knockdown in cultured primary porcine pinealocytes results in the decline of melatonin and arylalkylamine N-acetyltransferase (AANAT) mRNA levels by 29.2% and 72.2%, respectively, whereas Isl1 overexpression raised by 1.3-fold and 2.7-fold. In addition, the enhancing effect of norepinephrine (NE) on melatonin synthesis was abolished by Isl1 knockdown. The in vivo intracerebroventricular NE injections upregulate Isl1 mRNA and protein levels by about threefold and 4.5-fold in the porcine pineal gland. We then examined the changes in Isl1 expression in the pineal gland and global melatonin levels throughout the day. The results show that Isl1 protein level at 24:00 is 2.5-fold higher than that at 12:00, which is parallel to melatonin levels. We further found that Isl1 increases the activity of AANAT promoter, and the effect of NE on Isl1 expression was blocked by an ERK inhibitor. Collectively, the results presented here demonstrate that Isl1 positively modulates melatonin synthesis by targeting AANAT, via the ERK signaling pathway of NE. These suggest that Isl1 plays important roles in maintaining the daily circadian rhythm.

We investigated the etiology and molecular mechanisms of bladder outlet obstruction (BOO). Transgenic (Tg) male mice overexpressing aromatase (Cyp19a1) under the ubiquitin C promoter in the estrogen-susceptible C57Bl/6J genetic background (AROM؉/ 6J) developed inguinal hernia by 2 months and severe BOO by 9 to 10 months, with 100% penetrance. These mice gradually developed uremia, renal failure, renal retention, and finally died. The BOO bladders were threefold larger than in age-matched wild-type (WT) males and were filled with urine on necropsy. Hypotrophic smooth muscle cells formed the thin detrusor urinae muscle, and collagen III accumulation contributed to the reduced compliance of the bladder. p-AKT and ER␣ expression were up-regulated and Pten expression was down-regulated in the BOO bladder urothelium. Expression of only ER␣ in the intradetrusor fibroblasts suggests a specific role of this estrogen receptor form in urothelial proliferation. Inactivation of Pten, which in turn activated the p-AKT pathway, was strictly related to the activation of the ER␣ pathway in the BOO bladders. Human relevance for these findings was provided by increased expression of p-AKT, PCNA, and ER␣ and decreased expression of PTEN in severe human BOO samples, compared with subnormal to mild samples. These findings clarify the involvement of estrogen excess and/or imbalance of the androgen/estrogen ratio in the molecular pathogenetic mechanisms of BOO and provide a novel lead into potential treatment strategies for BOO. A close interrelationship between lower urinary tract (bladder and urethra) symptoms, bladder outlet obstruction (BOO), and benign prostatic hyperplasia has been shown in aging men. 1-4 These symptoms, which include increased voiding frequency and urgency, nocturia, incomplete bladder emptying, hesitancy, weak stream, and straining, occur in mild to severe form in 50% to 85% of men over 50 years of age. 5-8 BOO, which reduces or prevents the flow of urine into the urethra, and urinary tract infection, bladder cancer, and incontinence comprise the major causes of lower urinary tract symptoms. 9,10 Congenital or acquired BOO can result in a stiff-walled, fibrotic bladder with low capacity, high pressure, and noncompliance, which may ultimately damage the kidneys. 10 The incidence of lower urinary tract symp-

MicroRNAs, including microRNA‐7 (miR‐7), are important modulators of numerous gene expressions and the related biological processes. Melatonin is a key hormone regulating daily and seasonal rhythms, in which a variety of positive and negative regulatory factors, such as norepinephrine (NE) and leptin, are involved. However, the interactions among these factors and the mechanisms remain to be elucidated. The aims of the present study were to identify the functions and the related mechanisms of miR‐7 in regulating melatonin synthesis and secretion through in vitro and in vivo experiments in pineal gland of pigs, which is an important animal model for agricultural and biomedical studies. Our results firstly show that miR‐7 is specifically expressed in porcine pinealocytes and negatively regulates melatonin synthesis. The further functional studies show that the dynamic expression levels of miR‐7 are contrary to the melatonin levels throughout the day, and the forced inhibition of endogenous miR‐7 in porcine pinealocytes sharply increases arylalkylamine N‐acetyltransferase (AANAT) expression by 80.0% (P = 0.0031) and melatonin levels by 81.0% (P = 0.0421), whereas miR‐7 over‐expression down‐regulates AANAT expression by 38.6% (P = 0.0004) and melatonin levels by 37.6% (P = 0.0212). In addition, the miR‐7 expression is up‐regulated by leptin through the JAK/STAT3 signaling pathway, and the in vivo intracerebroventricular injection of leptin increases miR‐7 expression by 80.0% (P = 0.0044) in porcine pineal glands and reduces melatonin levels by 57.1% (P = 0.0060) compared with the controls. This functional inhibition of melatonin synthesis by miR‐7 is accomplished by its binding to the 3′‐UTR of Raf1. Further, our results demonstrate that the RAF1/MEK/ERK signaling pathway mediates NE‐induced AANAT expression, whereas leptin attenuates NE's function through miR‐7. Taken together, the results demonstrated that leptin activates the JAK/STAT3 signaling pathway to increase the expression of miR‐7, which acts as a negative regulatory molecule inhibiting NE‐activated RAF1/MEK/ERK signaling pathway by targeting Raf1, resulting in decreased AANAT expression and melatonin synthesis. These findings suggest that miR‐7 is a novel negative regulator of melatonin synthesis and links leptin‐ and NE‐mediated signaling pathways in porcine pineal glands, which will contribute to our understanding in the establishment of the biological rhythms resulting from melatonin.

Zearalenone (ZEA) acts as an environmental endocrine disruptor (EED) to cause health detriments. miRNAs were reported to influence the synthesis and secretion of pituitary hormones. However, the interactions between ZEA and miRNAs and related mechanisms remain unclear. The aims of this study were to determine whether and how miR-7 affects animal reproduction by its interactions with ZEA in the pig pituitary, which is sensitive to ZEA and has been used as an important animal model in medical research. Expressions of miRNA were detected by real-time PCR, in situ hybridization, and immunohistochemistry. The effects of ZEA, miR-7, and their interactions in the pituitary gland were identified by using an ovariectomized pig model, transfecting miR-7 mimics and inhibitor, radioimmunoassay, luciferase reporter assay, and Western blotting. The ZEA dosage was 7.5 mg/kg body weight in vivo and 1 μM in vitro. Our results demonstrate miR-7 acts to regulate gonadotropin synthesis and secretion. Furthermore, we found that ZEA leads to reproductive defects by enhancing miR-7 expression, which subsequently inhibits FSH synthesis and secretion. In vitro and in vivo experiments revealed that the effects of ZEA rely on G protein-coupled estrogen receptor 1, and miR-7 functions by mediating ZEA signaling pathway and targeting the Finkel-Biskis-Jinkins murine osteosarcoma viral oncogene homolog (FOS) gene. These findings show that miRNAs are key intrinsic factors regulating pituitary gonadotropins by mediating EED signaling in pituitary glands, and the actions of miRNAs and EEDs should be seriously considered in related studies about medical practice and animal production.