Inhibition of Connective Tissue Growth Factor Expression in Adult Retinal Pigment Epithelial-19 Cells by Blocking Yes-Associated Protein/Transcriptional Coactivator with PDZ-Binding Motif Activity.

Purpose: To investigate the effect of yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) on connective tissue growth factor (CTGF) expression in adult retinal pigment epithelial (ARPE)-19 cells. We also studied the inhibitory effect of K-975, a new pan-transcriptional enhanced associate domain (TEAD) inhibitor, and luteolin, a plant-derived flavonoid on CTGF expression. Methods: ARPE-19 cells were transfected with either YAP or TAZ overexpression plasmid or treated with transforming growth factor (TGF)-ß2. The cells were cultured either with or without K-975 or luteolin. The expression of YAP, TAZ, and CTGF was examined using real-time PCR. Results: ARPE-19 cells overexpressing YAP or TAZ exhibited significantly increased CTGF expression. This increase was attenuated by K-975 or luteolin alone. TGF-ß2 treatment significantly raised the expression of not just YAP and TAZ, but also CTGF in ARPE-19 cells. TGF-ß2 treatment-enhanced CTGF expression was considerably lowered by the addition of K-975 or luteolin. Conclusions: Overexpression of YAP or TAZ and treatment with TGF-ß2 led to an increase in the expression of CTGF in ARPE-19 cells. These increases were attenuated by treatment with K-975 and luteolin. These findings suggest that YAP and TAZ may be related to the expression of CTGF in ARPE-19 cells and that K-975 and luteolin can be explored as potential therapeutic agents for preventing CTGF production in vitreoretinal fibrosis.

Modification of cardiac disease by transgenically altered histone deacetylase 6.

Histone deacetylase 6 (HDAC6) is known to deacetylate amino acid lysine in alpha-tubulin. However, the functional role of HDAC6 in the progression of cardiac disease remains uncertain. The functional role of HDAC6 in the hearts was examined using transgenic (TG) mice expressing either human wild-type HDAC6, deacetylase inactive HDAC6 (HDAC6H216A, H611A), and human HDAC6 replaced all serine or threonine residues with aspartic acid at N-terminal 1- 43 amino acids (HDAC6NT-allD) specifically in the hearts. Overexpression of wild-type HDAC6 significantly reduced acetylated tubulin levels, and overexpression of HDAC6H216A, H611A significantly increased it in the mouse hearts. Detectable acetylated tubulin disappeared in HDAC6NT-allD TG mouse hearts. Neither histological alteration nor alteration of cardiac function was observed in the HDAC6 TG mouse hearts. To analyze the role of HDAC6 and acetylated tubulin in disease conditions, we examined HDAC6 in isoprenaline-induced hypertrophy or pressure-overload hypertrophy (TAC). No obvious alteration in the heart weight/body weight ratio or gene expressions of hypertrophic markers between NTG and HDAC6NT-allD mice was observed following treatment with isoprenaline. In contrast, a marked reduction in the shortening fraction and dilated chamber dilatation was detected in the HDAC6NT-allD TG mouse hearts 2 weeks after TAC. A sustained low level of acetylated tubulin and acetylated cortactin was observed in the TAC HDAC6NT-allD TG mouse hearts. Cardiac HDAC6 activity that can regulate acetylated levels of tubulin and cortactin may be critical factors involved in cardiac disease such as pressure-overload hypertrophy.

Mizagliflozin, a selective SGLT1 inhibitor, improves vascular cognitive impairment in a mouse model of small vessel disease.

Previously, we showed that sodium/glucose cotransporter 1 (SGLT1) participates in vascular cognitive impairment in small vessel disease. We hypothesized that SGLT1 inhibitors can improve the small vessel disease induced-vascular cognitive impairment. We examined the effects of mizagliflozin, a selective SGLT1 inhibitor, and phlorizin, a non-selective SGLT inhibitor, on vascular cognitive impairment in a mouse model of small vessel disease. Small vessel disease was created using a mouse model of asymmetric common carotid artery surgery (ACAS). Two and/or 4 weeks after ACAS, all experiments were performed. Cerebral blood flow (CBF) was decreased in ACAS compared with sham-operated mice. Phlorizin but not mizagliflozin reversed the decreased CBF of ACAS mice. Both mizagliflozin and phlorizin reversed the ACAS-induced decrease in the latency to fall in a wire hang test of ACAS mice. Moreover, they reversed the ACAS-induced longer escape latencies in the Morris water maze test of ACAS mice. ACAS increased SGLT1 and proinflammatory cytokine gene expressions in mouse brains and phlorizin but not mizagliflozin normalized all gene expressions in ACAS mice. Hematoxylin/eosin staining demonstrated that they inhibited pyknotic cell death in the ACAS mouse hippocampus. In PC12HS cells, IL-1ß increased SGLT1 expression and decreased survival rates of cells. Both mizagliflozin and phlorizin increased the survival rates of IL-1ß-treated PC12HS cells. These results suggest that mizagliflozin and phlorizin can improve vascular cognitive impairment through the inhibition of neural SGLT1 and phlorizin also does so through the improvement of CBF in a mouse model of small vessel disease.

Chronic HDAC6 Activation Induces Atrial Fibrillation Through Atrial Electrical and Structural Remodeling in Transgenic Mice.

Atrial fibrillation (AF) is a relatively common complication of hypertension. Chronic hypertension induces cardiac HDAC6 catalytic activity. However, whether HDAC6 activation contributes to hypertension-induced AF is still uncertain. We examined whether chronic cardiac HDAC6 activation-induced atrial remodeling, leading to AF induction.The HDAC6 constitutively active transgenic (TG) (HDAC6 active TG) mouse overexpressing the active HDAC6 protein, specifically in cardiomyocytes, was created to examine the effects of chronic HDAC6 activation on atrial electrical and structural remodeling and AF induction in HDAC6 active TG and non-transgenic (NTG) mice. Left atrial burst pacing (S1S1 = 30 msec) for 15-30 sec significantly increased the frequency of sustained AF in HDAC6 active-TG mice compared with NTG mice. Left steady-state atrial pacing (S1S1 = 80 msec) decreased the atrial conduction velocity in isolated HDAC6 active TG compared with NTG mouse atria. The atrial size was similar between HDAC6 active TG and NTG mice. In contrast, atrial interstitial fibrosis increased in HDAC6 active TG compared with that of NTG mouse atria. While protein expression levels of both CX40 and CX43 were similar between HDAC6 active TG and NTG mouse atria, a heterogeneous distribution of CX40 and CX43 occurred in HDAC6 active-TG mouse atria but not in NTG mouse atria. Gene expression of interleukin 6 increased in HDAC6 active TG compared with NTG mouse atria.Chronic cardiac HDAC6 activation induced atrial electrical and structural remodeling, and sustained AF. Hypertension-induced cardiac HDAC6 catalytic activity may play important roles in the development of AF.

Cardiac dopamine D1 receptor triggers ventricular arrhythmia in chronic heart failure.

Pathophysiological roles of cardiac dopamine system remain unknown. Here, we show the role of dopamine D1 receptor (D1R)-expressing cardiomyocytes (CMs) in triggering heart failure-associated ventricular arrhythmia. Comprehensive single-cell resolution analysis identifies the presence of D1R-expressing CMs in both heart failure model mice and in heart failure patients with sustained ventricular tachycardia. Overexpression of D1R in CMs disturbs normal calcium handling while CM-specific deletion of D1R ameliorates heart failure-associated ventricular arrhythmia. Thus, cardiac D1R has the potential to become a therapeutic target for preventing heart failure-associated ventricular arrhythmia.

Physiologic changes in serotonin concentrations in breast milk during lactation.

OBJECTIVES: Serotonin (5-hydroxytryptamine; 5-HT) plays an important role in milk volume homeostasis in the mammary glands during lactation, and 5-HT in milk also may affect infant development. The aim of this study was to investigate changes in 5-HT concentration in breast milk according to the duration of lactation and evaluate whether the 5-HT concentration varied before and after nursing. METHODS: Healthy nursing Japanese women who had a natural delivery or underwent a cesarean delivery at Iwate Medical University Hospital were included in this study. RESULTS: The mean 5-HT concentration in milk was obtained from multiparous mothers 6 to 7 d after delivery (colostrum) and was significantly higher compared with primiparous mothers (24.3 ± 2.63 versus 18.5 ± 2.60 ng/mL). Additionally, mean 5-HT concentration increased with increasing lactation duration in primiparous women (colostrum: 18.5 ± 2.60; 1 mo postdelivery: 19.8 ± 2.46; 3 mo postdelivery: 22.7 ± 2.55 ng/mL); in particular, the mean 5-HT concentration in breast milk 3 mo after delivery was significantly higher than in colostrum. The mean 5-HT concentrations in breast milk in primiparous mothers immediately before nursing, 1 to 2 h after nursing, and immediately before the next nursing event were 23.6 ± 1.48, 22.82 ± 1.65, and 21.84 ± 1.31 ng/mL, respectively; mean 5-HT concentrations in multiparous women were 25.4 ± 1.65, 23.6 ± 2.20, or 22.4 ± 2.09 ng/mL, respectively. There was no significant difference in 5-HT concentrations at each time point between the groups. CONCLUSION: This information may be useful in determining the role of 5-HT in breast milk on infant development and growth.

Topical administration of a ROCK inhibitor prevents anterior subcapsular cataract induced by UV-B irradiation.

The deposition of extracellular matrix (ECM)-which is mainly composed of type I collagen-in anterior subcapsular cataracts (ASCs) during epithelial-to-mesenchymal transition (EMT) of lens epithelial cells (LECs) decreases visual function. Transforming growth factor (TGF)-ß is a key factor in the induction of EMT in LECs. Although Rho kinase (ROCK) plays an important role in EMT induced by TGF-ß, it is unknown whether ROCK inhibition affects type I collagen expression in TGF-ß-stimulated LECs and ASC formation. This was investigated in the present study both in vitro using human lens epithelium (HLE)-B3 cells and in vivo using mice with ultraviolet radiation (UVR)-B-induced cataracts. We found that TGF-ß2 increased type I collagen mRNA expression in HLE-B3 cells; this was inhibited in a dose-dependent manner by treatment with the ROCK inhibitor Y-27632. UVR-B exposure caused ASC formation in mice. A histopathological examination revealed that LECs in the anterior subcapsular area were flattened and multi-layered, and had a spindle shape in cross section. Immunohistochemical analysis revealed the presence of α-smooth muscle actin and type I collagen around these flattened LECs; these opacities were reduced by topical instillation of Y-27632. These findings suggest that suppression of TGF-ß signaling in LECs by topical application of a ROCK inhibitor can prevent the formation of ASCs.

Chronic Pressure Overload Induces Cardiac Hypertrophy and Fibrosis via Increases in SGLT1 and IL-18 Gene Expression in Mice.

Increased gene expression levels of sodium-glucose cotransporter 1 (SGLT1) are associated with hypertrophic and ischemic cardiomyopathy. However, it remains unclear whether chronic pressure overload increases SGLT1 expression, which in turn induces hypertrophic cardiomyopathy. We hypothesized that pressure overload could increase SGLT1 gene expression, leading to the development of hypertrophic cardiomyopathy.To create pressure overload-induced cardiomyopathy, transverse aortic constriction (TAC) was performed in SGLT1-deficient (SGLT1-/-) and wild-type (WT) mice. Six weeks after surgery, all mice were investigated. We observed a reduction of left ventricular fractional shortening and left ventricular dilatation in TAC-operated WT but not in TAC-operated SGLT1-/- mice. SGLT1, interleukin 18, connective tissue growth factor, and collagen type 1 gene expression levels were increased in TAC-operated WT mouse hearts compared with that of sham-operated WT mouse hearts. Moreover, heart/body weight ratio and ventricular interstitial fibrosis were increased in TAC-operated WT mice compared with that of sham-operated WT mice. Interestingly, these factors did not increase in TAC-operated SGLT1-/- mice compared with that of sham-operated WT and SGLT1-/- mice. Phenylephrine, an adrenergic α1 receptor agonist, caused cardiomyocyte hypertrophy in neonatal WT mouse hearts to a significantly larger extent than in neonatal SGLT1-/- mouse hearts.In conclusion, the results indicate that chronic pressure overload increases SGLT1 and IL-18 gene expressions, leading to the development of hypertrophic cardiomyopathy. These results make SGLT1 a potential candidate for the therapeutic target for hypertension-induced cardiomyopathy.

Bcl-2-associated athanogene 3 (BAG3) is an enhancer of small heat shock protein turnover via activation of autophagy in the heart.

Bcl-2-associated athanogene 3 (BAG3) is strongly expressed in both cardiac and skeletal muscle. A recent study showed that BAG3 may play a protective role in muscles. Little is known, however, regarding the detailed role of BAG3 in cardiac muscle. To better understand the functional role of cardiac BAG3 in the heart, we generated transgenic (TG) mice that overexpress BAG3. A decrease in fractional shortening, and the induction of cardiac atrial natriuretic peptide, were observed in BAG3 TG mice. Moreover, a marked reduction in the protein level of small HSPs was detected in BAG3 TG mouse hearts. We analyzed the cardiac small HSP levels when either the ubiquitin-proteasome system (UPS) or the autophagy system (AS) was inhibited in BAG3 TG mice. The protein turnovers of small HSPs by the AS were activated in BAG3 TG mouse hearts. Thus, BAG3 is critical for the protein turnover of small HSPs via activation of autophagy in the heart.

Analysis of serotonin concentrations in human milk by high-performance liquid chromatography with fluorescence detection.

Serotonin (5-hydroxytryptamine, 5-HT) plays an important role in milk volume homeostasis in the mammary gland during lactation; 5-HT in milk may also affect infant development. However, there are few reports on 5-HT concentrations in human breast milk. To address this issue, we developed a simple method based on high-performance liquid chromatography with fluorescence detection (HPLC-FD) for measuring 5-HT concentrations in human breast milk. Breast milk samples were provided by four healthy Japanese women. Calibration curves for 5-HT in each sample were prepared with the standard addition method between 5 and 1000 ng/ml, and all had correlation coefficients >0.999. The recovery of 5-HT was 96.1%-101.0%, with a coefficient of variation of 3.39%-8.62%. The range of 5-HT concentrations estimated from the calibration curves was 11.1-51.1 ng/ml. Thus, the HPLC-FD method described here can effectively extract 5-HT from human breast milk with high reproducibility.

Serotonin suppresses ß-casein expression via PTP1B activation in human mammary epithelial cells.

Serotonin (5-hydroxytriptamine, 5-HT) has an important role in milk volume homeostasis within the mammary gland during lactation. We have previously shown that the expression of ß-casein, a differentiation marker in mammary epithelial cells, is suppressed via 5-HT-mediated inhibition of signal transduction and activator of transcription 5 (STAT5) phosphorylation in the human mammary epithelial MCF-12A cell line. In addition, the reduction of ß-casein in turn was associated with 5-HT7 receptor expression in the cells. The objective of this study was to determine the mechanisms underlying the 5-HT-mediated suppression of ß-casein and STAT5 phosphorylation. The ß-casein level and phosphorylated STAT5 (pSTAT5)/STAT5 ratio in the cells co-treated with 5-HT and a protein kinase A (PKA) inhibitor (KT5720) were significantly higher than those of cells treated with 5-HT alone. Exposure to 100 µM db-cAMP for 6 h significantly decreased the protein levels of ß-casein and pSTAT5 and the pSTAT5/STAT5 ratio, and significantly increased PTP1B protein levels. In the cells co-treated with 5-HT and an extracellular signal-regulated kinase1/2 (ERK) inhibitor (FR180294) or Akt inhibitor (124005), the ß-casein level and pSTAT5/STAT5 ratio were equal to those of cells treated with 5-HT alone. Treatment with 5-HT significantly induced PTP1B protein levels, whereas its increase was inhibited by KT5720. In addition, the PTP1B inhibitor sc-222227 increased the expression levels of ß-casein and the pSTAT5/STAT5 ratio. Our observations indicate that PTP1B directly regulates STAT5 phosphorylation and that its activation via the cAMP/PKA pathway downstream of the 5-HT7 receptor is involved in the suppression of ß-casein expression in MCF-12A cells.

Inhibitory effect of fluvoxamine on ß-casein expression via a serotonin-independent mechanism in human mammary epithelial cells.

Selective serotonin reuptake inhibitors (SSRIs) are widely used as a first-line therapy in postpartum depression. The objective of this study was to determine the mechanism underlying the inhibitory effects of the SSRI, fluvoxamine, on ß-casein expression, an indicator of lactation, in MCF-12A human mammary epithelial cells. Expression levels of serotonin (5-hydroxytryptamine; 5-HT) transporter, an SSRI target protein, and tryptophan hydroxylase 1, a rate-limiting enzyme in 5-HT biosynthesis, were increased in MCF-12A cells by prolactin treatment. Treatment with 1 µM fluvoxamine for 72 h significantly decreased protein levels of ß-casein and phosphorylated signal transducer and activator transcription 5 (pSTAT5). Extracellular 5-HT levels were significantly increased after exposure to 1 µM fluvoxamine, in comparison with those of untreated and vehicle-treated cells; however, extracellular 5-HT had little effect on the decrease in ß-casein expression. Expression of glucose-related protein 78/binding immunoglobulin protein, a regulator of endoplasmic reticulum (ER) stress, was significantly increased after treatment with 1 µM fluvoxamine for 48 h. Exposure to tunicamycin, an inducer of ER stress, also decreased expression of ß-casein and pSTAT5 in a manner similar to fluvoxamine. Our results indicate that fluvoxamine suppresses ß-casein expression in MCF-12A cells via inhibition of STAT5 phosphorylation caused by induction of ER stress. Further studies are required to confirm the effect of fluvoxamine on the function of mammary epithelial cells.

Serotonin regulates ß-casein expression via 5-HT7 receptors in human mammary epithelial MCF-12A cells.

We previously reported that serotonin (5-hydroxytryptamine; 5-HT) suppresses ß-casein expression, a differentiation marker in mammary epithelial cells, via inhibition of the signal transducer and activator of transcription 5 (STAT5) phosphorylation in the human mammary epithelial cell line, MCF-12A. In this study, we investigated the expression pattern of the different 5-HT receptor subtypes in MCF-12A cells, and identified the receptors involved in 5-HT-mediated suppression of ß-casein protein expression. ß-Casein mRNA expression was inhibited by 30 µM 5-HT in a time-dependent manner. Treatment with 30 µM 5-HT for 72 h decreased ß-casein protein levels and STAT5 phosphorylation (pSTAT5). The cells expressed four 5-HT receptors subtypes (5-HTR1D, 2B, 3A, and 7) at the mRNA and protein level, and their expression was elevated by prolactin (PRL) treatment. Additionally, the mRNA levels of 5-HTR1D and 5-HTR7 were significantly higher than the other 5-HT receptors in the cells. Tryptophan hydroxylase 1 mRNA was detectable in the cells in the absence of PRL, and PRL treatment significantly increased its expression. ß-Casein and pSTAT5/STAT5 levels in the cells co-treated with 5-HT and a selective 5-HTR1D inhibitor, BRL15572, were equal to those observed in cells treated with 5-HT alone. However, in the cells co-treated with 5-HT and a selective 5-HTR7 inhibitor, SB269970, ß-casein and pSTAT5/STAT5 levels increased in a SB269970 concentration-dependent manner. In conclusion, we showed that 5-HT regulates ß-casein expression via 5-HTR7 in MCF-12A human mammary epithelial cells.

Upregulation of inorganic pyrophosphatase 1 as a JNK phosphatase in hypothyroid embryonic chick cerebellum.

AIM: Thyroid hormones play important roles in vertebrate neuronal development and differentiation. In our previous study, we showed that fetal thyroid dysfunction led to impaired social behaviors of hatchlings on post-hatch day 3, as well as to impaired learning and memory determined by the imprinting preference. However, little is known about the mechanisms underlying the direct adverse effects of fetal thyroid dysfunction on neuronal development. MATERIALS AND METHODS: We used a chick embryo as a fetal model to investigate the effects of prenatal exposure to antithyroid drugs on neuronal development in the chick cerebellum. Methimazole (MMI) at a dose of 20µmol/egg was administered to eggs on day 14, while the control was given only a vehicle. In order to address the underlying mechanisms of the impaired behavior, proteomic approaches were employed in the chick cerebellum two days after MMI treatment. KEY FINDINGS: In this experiment, we found that inorganic pyrophosphatase 1 (PPA1) was upregulated in the chick cerebellum treated with MMI, and we confirmed this upregulation of PPA1 by Western blot analysis as well as by RT-PCR analysis. Concomitant with the upregulation of PPA1, a marked reduction in JNK activity, as well as of phospho-JNK level, was detected in the MMI-treated chick cerebellum. SIGNIFICANCE: Since PPA1 can dephosphorylate JNK, these results suggest that the upregulation of PPA1 during neuronal development in the hypothyroid chick cerebellum may lead to impaired social behaviors as well as to impaired learning and memory via JNK dephosphorylation and inactivation in the chick cerebellum.

Effect of astaxanthin on cataract formation induced by glucocorticoids in the chick embryo.

PURPOSE: To examine whether astaxanthin (AST) prevent the cataract formation induced by glucocorticoid in chick embryo. MATERIALS AND METHODS: Hydrocortisone hemisuccinate sodium (HC) (0.5 µmol/egg) was administered directly into the air chamber in the egg shell of chick embryo day 15. The eggs were then kept in an incubator at same conditions and administered 100 µL of 50 (HC + AST50 group), 80 (HC + AST80 group), 100 (HC + AST100 group) mg/mL of AST solutions dissolved in dimethyl sulfoxide (DMSO) 3 h after administration of HC. In addition, non-HC treated group (treated with physiological saline without HC and 100 µL of DMSO), HC-alone group (treated with 0.5 µmol of HC and 100 µL of DMSO), and AST100 group (treated with physiological saline without HC and 100 µL of DMSO) were also incorporated. After 48 h of treatment, lenses were removed from embryo and classified into five stages according to developed opacity. The amounts of reduced glutathione in the lenses and the blood glucose levels were measured. RESULTS: The average scores of lens opacitiy were 2.63 ± 1.02 nmol/lens (HC-alone), 2.78 ± 0.97 nmol/lens (HC + AST50), 2.22 ± 1.20 nmol/lens (HC + AST80) and 1.84 ± 0.83 nmol/lens (HC + AST100; p < 0.05), respectively. Administration of AST decreased the lens opacity dose-dependently. The amounts of reduced glutathione in lenses were 11.6 ± 2.8 nmol/lens (HC-alone), 11.3 ± 2.7 nmol/lens (HC + AST50), 13.4 ± 2.4 nmol/lens (HC + AST80) and 13.7 ± 3.1 nmol/lens (HC + AST100; p < 0.05), respectively. Higher levels of AST prevented loss of reduced glutathione from the lens. CONCLUSION: These findings support that AST protects glucocorticoid-induced cataract in chick embryo.

Arf6 guanine nucleotide exchange factor cytohesin-2 binds to CCDC120 and is transported along neurites to mediate neurite growth.

The mechanism of neurite growth is complicated, involving continuous cytoskeletal rearrangement and vesicular trafficking. Cytohesin-2 is a guanine nucleotide exchange factor for Arf6, an Arf family molecular switch protein, controlling cell morphological changes such as neuritogenesis. Here, we show that cytohesin-2 binds to a protein with a previously unknown function, CCDC120, which contains three coiled-coil domains, and is transported along neurites in differentiating N1E-115 cells. Transfection of the small interfering RNA (siRNA) specific for CCDC120 into cells inhibits neurite growth and Arf6 activation. When neurites start to extend, vesicles containing CCDC120 and cytohesin-2 are transported in an anterograde manner rather than a retrograde one. As neurites continue extension, anterograde vesicle transport decreases. CCDC120 knockdown inhibits cytohesin-2 localization into vesicles containing CCDC120 and diffuses cytohesin-2 in cytoplasmic regions, illustrating that CCDC120 determines cytohesin-2 localization in growing neurites. Reintroduction of the wild type CCDC120 construct into cells transfected with CCDC120 siRNA reverses blunted neurite growth and Arf6 activity, whereas the cytohesin-2-binding CC1 region-deficient CCDC120 construct does not. Thus, cytohesin-2 is transported along neurites by vesicles containing CCDC120, and it mediates neurite growth. These results suggest a mechanism by which guanine nucleotide exchange factor for Arf6 is transported to mediate neurite growth.

Serotonin suppresses ß-casein expression via inhibition of the signal transducer and activator of transcription 5 (STAT5) protein phosphorylation in human mammary epithelial cells MCF-12A.

Serotonin (5-hydroxytryptamine; 5-HT) has an important physiological role in controlling lactation, namely, milk volume homeostasis, within mammary glands. The objectives of this study were to evaluate whether exogenous 5-HT can suppress ß-casein expression, a differentiation marker, produced in human mammary epithelial cells, and to determine whether 5-HT can attenuate ß-casein signaling via the prolactin (PRL) receptor (PRLr) and Janus kinase 2/signal transducer and activator of transcription 5 (STAT5) pathway. PRL treatment increased the mRNA level of ß-casein in the MCF-12A human mammary epithelial cell line, and the highest level occurred at days 7 and 14 of culture. In contrast, PRLr expression was not affected significantly by PRL treatment. PRL treatment in MCF-12A cells increased levels of ß-casein and phosphorylated STAT5 (pSTAT5) proteins in a concentration-dependent manner, with a slight increase of STAT5 protein. ß-Casein expression was inhibited by 0.1 mM 5-HT in a time-dependent manner. Additionally, treatment with 0.1 mM 5-HT for 72 h decreased protein levels of ß-casein and pSTAT5, with a slight decrease in STAT5 levels. These results suggest that exogenous 5-HT can inhibit STAT5 phosphorylation, resulting in a decrease in ß-Casein expression. In conclusion, we showed that exogenous 5-HT decreased ß-casein expression in MCF-12A human mammary epithelial cells, and that 5-HT was responsible for inhibiting phosphorylation of STAT5, resulting in a decline in lactational function.

Cardiac overexpression of constitutively active Galpha q causes angiotensin II type1 receptor activation, leading to progressive heart failure and ventricular arrhythmias in transgenic mice.

BACKGROUND: Transgenic mice with transient cardiac expression of constitutively active Galpha q (Gαq-TG) exhibt progressive heart failure and ventricular arrhythmias after the initiating stimulus of transfected constitutively active Gαq becomes undetectable. However, the mechanisms are still unknown. We examined the effects of chronic administration of olmesartan on heart failure and ventricular arrhythmia in Gαq-TG mice. METHODOLOGY/PRINCIPAL FINDINGS: Olmesartan (1 mg/kg/day) or vehicle was chronically administered to Gαq-TG from 6 to 32 weeks of age, and all experiments were performed in mice at the age of 32 weeks. Chronic olmesartan administration prevented the severe reduction of left ventricular fractional shortening, and inhibited ventricular interstitial fibrosis and ventricular myocyte hypertrophy in Gαq-TG. Electrocardiogram demonstrated that premature ventricular contraction (PVC) was frequently (more than 20 beats/min) observed in 9 of 10 vehicle-treated Gαq-TG but in none of 10 olmesartan-treated Gαq-TG. The collected QT interval and monophasic action potential duration in the left ventricle were significantly shorter in olmesartan-treated Gαq-TG than in vehicle-treated Gαq-TG. CTGF, collagen type 1, ANP, BNP, and ß-MHC gene expression was increased and olmesartan significantly decreased the expression of these genes in Gαq-TG mouse ventricles. The expression of canonical transient receptor potential (TRPC) 3 and 6 channel and angiotensin converting enzyme (ACE) proteins but not angiotensin II type 1 (AT1) receptor was increased in Gαq-TG ventricles compared with NTG mouse ventricles. Olmesartan significantly decreased TRPC6 and tended to decrease ACE expressions in Gαq-TG. Moreover, it increased AT1 receptor in Gαq-TG. CONCLUSIONS/SIGNIFICANCE: These findings suggest that angiotensin II type 1 receptor activation plays an important role in the development of heart failure and ventricular arrhythmia in Gαq-TG mouse model of heart failure.