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Endometrial decidualization refers to a series of morphological changes and functional remodeling of the uterine endometrium to accept the embryo under the effect of estrogen and progesterone secreted by ovaries after ovulation. During decidualization, endometrial stromal cells (ESCs) proliferate and differentiate into decidual stromal cells, undergoing cytoskeletal rearrangement-mediated morphological changes and expressing decidualization markers, such as insulin-like growth factor-binding protein-1 and prolactin. Ras homology (Rho) proteins, a family of small G proteins, are well known as regulators of cellular morphology and involved in multiple other cellular processes. In this study, we found ras homolog family member B (RHOB) was the most significantly upregulated gene in the Rho protein family after the in vitro decidualization of human primary ESCs. RhoB expression was induced mainly by 3′,5′-cyclic adenosine 5′-monophosphate (cAMP) / protein kinase A (PKA) / cyclic adenosine monophosphate-response element binding protein signaling and partly by progesterone signaling. Knockdown of RhoB in ESCs greatly inhibited actin cytoskeletal rearrangement, cell morphological transformation, and upregulation of insulin-like growth factor-binding protein-1, suggesting an indispensable role of RhoB in decidualization. Mechanistically, the downstream target of RhoB was semaphorin3A (Sema3A), which mediated its signaling via interacting with the receptor, plexinA4. More importantly, decreased expression of RhoB, Sema3A, and plexinA4 were detected in deciduas from patients with unexplained spontaneous miscarriage. Collectively, our results indicate that RhoB/Sema3A/plexinA4 signaling plays a positive role in endometrial decidualization and relates to unexplained spontaneous miscarriage, which is worthy of further exploration so as to provide new insights into therapeutic strategies for pregnancy diseases associated with poor decidualization.

MicroRNAs (miRNAs) expressed in the hypothalamus are capable of regulating energy balance and peripheral metabolism by inhibiting translation of target messenger RNAs (mRNAs). Hypothalamic insulin resistance is known to precede that in the periphery, thus a critical unanswered question is whether central insulin resistance creates a specific hypothalamic miRNA signature that can be identified and targeted. Here we show that miR-1983, a unique miRNA, is upregulated in vitro in 2 insulin-resistant immortalized hypothalamic neuronal neuropeptide Y-expressing models, and in vivo in hyperinsulinemic mice, with a concomitant decrease of insulin receptor β subunit protein, a target of miR-1983. Importantly, we demonstrate that miR-1983 is detectable in human blood serum and that its levels significantly correlate with blood insulin and the homeostatic model assessment of insulin resistance. Levels of miR-1983 are normalized with metformin exposure in mouse hypothalamic neuronal cell culture. Our findings provide evidence for miR-1983 as a unique biomarker of cellular insulin resistance, and a potential therapeutic target for prevention of human metabolic disease.

Late-onset hypogonadism (LOH) syndrome, due to a partial lack of testosterone, decreases the quality of life of older men. Testosterone is mainly secreted by Leydig cells in the testes. Leydig cell transplantation is expected to be a promising alternative to conventional testosterone replacement therapy for LOH syndrome. We herein report a simple and robust protocol for directed differentiation of human induced pluripotent stem cells (hiPSCs) into Leydig-like cells by doxycycline-inducible overexpression of NR5A1 and treatment with a combination of 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP) and forskolin. The differentiated cells expressed the steroidogenic enzyme genes STAR, CYP11A1, CYP17A1, and HSD3B2 and the specific markers of adult Leydig cells HSD17B3, INSL3, and LHCGR. Furthermore, we confirmed the secretion of functional testosterone from the cells into the culture supernatant by a testosterone-sensitive cell proliferation assay. These findings showed that the hiPSCs were able to be differentiated into Leydig-like cells, supporting the expectation that hiPSC-derived Leydig-like cells can be novel tools for treating LOH syndrome.

Glucocorticoid causes hyperglycemia, which is common in patients with or without diabetes. Prolonged hyperglycemia can be experienced even after the discontinuation of glucocorticoid use. In the present study, we examined the time course of blood glucose level in hospital patients who received transient glucocorticoid treatment. In addition, the mechanism of prolonged hyperglycemia was investigated by using dexamethasone (Dexa)-treated mice and cultured cells. The blood glucose level in glucose tolerance tests, level of insulin and glucagon-like peptide 1 (GLP-1), and the activity of dipeptidyl peptidase 4 (DPP-4) were examined during and after Dexa loading in mice, with histone acetylation level of the promoter region. Mice showed prolonged hyperglycemia during and after transient Dexa loading accompanied by persistently lower blood GLP-1 level and higher activity of DPP-4. The expression level of Dpp-4 was increased in the mononuclear cells and the promoter region of Dpp-4 was hyperacetylated during and after the transient Dexa treatment. In vitro experiments also indicated development of histone hyperacetylation in the Dpp-4 promoter region during and after Dexa treatment. The upregulation of Dpp-4 in cultured cells was significantly inhibited by a histone acetyltransferase inhibitor. Moreover, the histone hyperacetylation induced by Dexa was reversible by treatment with a sirtuin histone deacetylase activator, nicotinamide mononucleotide. We identified persistent reduction in blood GLP-1 level with hyperglycemia during and after Dexa treatment in mice, associated with histone hyperacetylation of promoter region of Dpp-4. The results unveil a novel mechanism of glucocorticoid-induced hyperglycemia, and suggest therapeutic intervention through epigenetic modification of Dpp-4.

Octamer-binding transcription factor 4 (OCT4) regulates the pluripotency of stem cells and also plays important roles in granulosa cells growth, which is regulated by follicle-stimulating hormone (FSH). Thyroid hormone (TH) is important for the development and maturation of follicles and the maintenance of various endocrine functions. Although 3,5,3′-triiodothyronine (T3) enhances the effects of FSH on the regulation of the growth of granulosa cells and development of follicles, it is unclear whether and, if so, how TH combines with FSH to regulate OCT4 expression in granulosa cells during the preantral to early antral transition stage. Our results showed that T3 enhanced FSH-induced OCT4 expression. However, T3/FSH-induced cellular growth was reduced by OCT4 small interfering RNA. OCT4 knockdown significantly increased the number of apoptotic cell. Moreover, T3 combined with FSH to increase estrogen receptor β (ERβ) expression but did not significantly affect estrogen receptor α expression. ERβ knockdown dramatically decreased T3/FSH-induced OCT4 expression and cell development and increased cell apoptosis. The phosphoinositide 3-kinases/protein kinase B pathway was involved in hormones inducing OCT4 and ERβ expressions. Furthermore, the hormones regulating OCT4 and ERβ expressions were regulated by cytochrome P450 lanosterol 14a-demethylase (CYP51), a key enzyme in sterol and steroid biosynthesis. T3 and FSH cotreatment potentiated cellular development by upregulating OCT4 expression, which is mediated by CYP51 and ERβ. These regulatory processes are mediated by the phosphoinositide 3-kinase/protein kinase B signaling pathway. These findings suggest that OCT4 mediates the T3 and FSH-induced development of follicles.

In the liver, the sterol response element binding protein (SREBP) and the SREBP cleavage-activated protein (SCAP) complex upregulate cholesterol biosynthesis by gene induction of de novo cholesterol synthetic enzymes (Hmgcr, Cyp51, and Dhcr7). Insulin induced gene 1 (INSIG1) negatively regulates cholesterol biosynthesis by the inhibition of de novo cholesterol biosynthetic gene expression. In the ovary, cholesterol is de novo synthesized; however, the roles of SREBP and its regulators (SCAP and INSIG1) are not well understood. In this study, when immature mice were treated with gonadotropins (eCG followed by hCG), eCG induced and hCG maintained the expression of SREBP-1a, -2, and SCAP granulosa cells, whereas INSIG1 expression was dramatically downregulated after hCG injection. Downregulation of INSIG1 led to generate the SREBPs active form and translocate the SREBPs active form to nuclei. Inhibition of generation of the SREBPs active form by fatostatin or Scap siRNA in both in vivo and in vitro significantly decreased the expressions of de novo cholesterol biosynthetic enzymes, cholesterol accumulation, and progesterone (P4) production compared with the control group. Fatostatin treatment inhibited the ovulation and increased the formation of abnormal corpus luteum which trapped the matured oocyte in the corpus luteum; however, the phenomenon was abolished by P4 administration. The results showed that decreasing INSIG1 level after hCG stimulation activated SREBP-induced de novo cholesterol biosynthesis in granulosa cells of preovulatory follicles, which is essential for P4 production and the rupture of matured oocyte during ovulation process.

Studies suggest that HIV-1 invades the testis through initial permeation of the blood–testis barrier (BTB). The selectivity of the BTB to antiretroviral drugs makes this site a sanctuary for the virus. Little is known about how HIV-1 crosses the BTB and invades the testis. Herein, we used 2 approaches to examine the underlying mechanism(s) by which HIV-1 permeates the BTB and gains entry into the seminiferous epithelium. First, we examined if recombinant Tat protein was capable of perturbing the BTB and making the barrier leaky, using the primary rat Sertoli cell in vitro model that mimics the BTB in vivo. Second, we used HIV-1–infected Sup-T1 cells to investigate the activity of HIV-1 infection on cocultured Sertoli cells. Using both approaches, we found that the Sertoli cell tight junction permeability barrier was considerably perturbed and that HIV-1 effectively permeates the BTB by inducing actin-, microtubule-, vimentin-, and septin-based cytoskeletal changes in Sertoli cells. These studies suggest that HIV-1 directly perturbs BTB function, potentially through the activity of the Tat protein.

Transitional hypoglycemia in normal newborns occurs in the first 3 days of life and has clinical features consistent with hyperinsulinism. We found a lower threshold for glucose-stimulated insulin secretion from freshly isolated embryonic day (E) 22 rat islets, which persisted into the first postnatal days. The threshold reached the adult level by postnatal day (P) 14. Culturing P14 islets also decreased the glucose threshold. Freshly isolated P1 rat islets had a lower threshold for insulin secretion in response to 2-aminobicyclo-(2, 2, 1)-heptane-2-carboxylic acid, a nonmetabolizable leucine analog, and diminished insulin release in response to tolbutamide, an inhibitor of β-cell KATP channels. These findings suggested that decreased KATP channel function could be responsible for the lower glucose threshold for insulin secretion. Single-cell transcriptomic analysis did not reveal a lower expression of KATP subunit genes in E22 compared with P14 β cells. The investigation of electrophysiological characteristics of dispersed β cells showed that early neonatal and cultured cells had fewer functional KATP channels per unit membrane area. Our findings suggest that decreased surface density of KATP channels may contribute to the observed differences in glucose threshold for insulin release.

Pericytes regulate vascular development, stability, and quiescence; their dysfunction contributes to diabetic retinopathy. To explore the role of insulin receptors in pericyte biology, we created pericyte insulin receptor knockout mice (PIRKO) by crossing PDGFRβ-Cre mice with insulin receptor (Insr) floxed mice. Their neonatal retinal vasculature exhibited perivenous hypervascularity with venular dilatation, plus increased angiogenic sprouting in superficial and deep layers. Pericyte coverage of capillaries was unaltered in perivenous and periarterial plexi, and no differences in vascular regression or endothelial proliferation were apparent. Isolated brain pericytes from PIRKO had decreased angiopoietin-1 mRNA, whereas retinal and lung angiopoietin-2 mRNA was increased. Endothelial phospho-Tie2 staining was diminished and FoxO1 was more frequently nuclear localized in the perivenous plexus of PIRKO, in keeping with reduced angiopoietin-Tie2 signaling. Silencing of Insr in human brain pericytes led to reduced insulin-stimulated angiopoietin-1 secretion, and conditioned media from these cells was less able to induce Tie2 phosphorylation in human endothelial cells. Hence, insulin signaling in pericytes promotes angiopoietin-1 secretion and endothelial Tie2 signaling and perturbation of this leads to excessive vascular sprouting and venous plexus abnormalities. This phenotype mimics elements of diabetic retinopathy, and future work should evaluate pericyte insulin signaling in this disease.

Endometriosis is characterized by inflammation and fibrotic changes. Our previous study using a mouse model showed that proinflammatory factors present in peritoneal hemorrhage exacerbated inflammation in endometriosis-like grafts, at least in part through the activation of prostaglandin (PG) E2 receptor and protease-activated receptor (PAR). In addition, menstruation-related factors, PGE2 and thrombin (P/T), a PAR1 agonist induced epithelial-mesenchymal transition (EMT) of endometrial cells under hypoxia. However, the molecular mechanisms by which P/T induce development of endometriosis have not been fully characterized. To investigate the effects of P/T, RNA extracted from endometrial stromal cells (ESCs) treated with P/T were subjected to RNA sequence analysis, and identified activin A, FOS, and GATA2 as upregulated genes. Activin A increased the expression of connective tissue growth factor (CTGF) and mesenchymal marker genes in ESCs. CTGF induced the expression of fibrosis marker type I collagen, fibronectin, and α-smooth muscle actin (αSMA), indicating fibroblast to myofibroblast transdifferentiation (FMT) of ESCs. In addition, activin A, FOS, GATA2, CTGF, and αSMA were localized in endometriosis lesions. Taken together, our data show that P/T induces changes resembling EMT and FMT in ectopic ESCs derived from retrograde menstruation, and that these are associated with fibrotic changes in the lesions. Pharmacological means that block P/T-induced activin A and CTGF signaling may be strategies to inhibit fibrosis in endometriotic lesions.

FOS, a subunit of the activator protein-1 (AP-1) transcription factor, has been implicated in various cellular changes. In the human ovary, the expression of FOS and its heterodimeric binding partners JUN, JUNB, and JUND increases in periovulatory follicles. However, the specific role of the FOS/AP-1 remains elusive. The present study determined the regulatory mechanisms driving the expression of FOS and its partners and functions of FOS using primary human granulosa/lutein cells (hGLCs). Human chorionic gonadotropin (hCG) induced a biphasic increase in the expression of FOS, peaking at 1 to 3 hours and 12 hours. The levels of JUN proteins were also increased by hCG, with varying expression patterns. Coimmunoprecipitation analyses revealed that FOS is present as heterodimers with all JUN proteins. hCG immediately activated protein kinase A and p42/44MAPK signaling pathways, and inhibitors for these pathways abolished hCG-induced increases in the levels of FOS, JUN, and JUNB. To identify the genes regulated by FOS, high-throughput RNA sequencing was performed using hGLC treated with hCG ± T-5224 (FOS inhibitor). Sequencing data analysis revealed that FOS inhibition affects the expression of numerous genes, including a cluster of genes involved in the periovulatory process such as matrix remodeling, prostaglandin synthesis, glycolysis, and cholesterol biosynthesis. Quantitative PCR analysis verified hCG-induced, T-5224-regulated expression of a selection of genes involved in these processes. Consistently, hCG-induced increases in metabolic activities and cholesterol levels were suppressed by T-5224. This study unveiled potential downstream target genes of and a role for the FOS/AP-1 complex in metabolic changes and cholesterol biosynthesis in granulosa/lutein cells of human periovulatory follicles.

Vasoinhibin is an endogenous prolactin (PRL) fragment with profibrinolytic, antivasopermeability, and antiangiogenic effects. The fact that blood clotting, vascular permeability, and angiogenesis are functionally linked during the wound-healing process led us to investigate whether thrombin, a major protease in tissue repair, generates vasoinhibin. Here, we have incubated human PRL with thrombin and analyzed the resulting proteolytic products by Western blot, mass spectrometry, high-performance liquid chromatography purification, recombinant production, and bioactivity. We unveil a main thrombin cleavage site at R48-G49 that rapidly (

Gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus play a key role in the regulation of reproductive function. In this study, we sought an efficient method for generating GnRH neurons from human embryonic and induced pluripotent stem cells (hESC and hiPSC, respectively). First, we found that exposure of primitive neuroepithelial cells, rather than neuroprogenitor cells, to fibroblast growth factor 8 (FGF8), was more effective in generating GnRH neurons. Second, addition of kisspeptin to FGF8 further increased the efficiency rates of GnRH neurogeneration. Third, we generated a fluorescent marker mCherry labeled human embryonic GnRH cell line (mCh-hESC) using a CRISPR-Cas9 targeting approach. Fourth, we examined physiological characteristics of GnRH (mCh-hESC) neurons: similar to GnRH neurons in vivo, they released the GnRH peptide in a pulsatile manner at ~60 min intervals; GnRH release increased in response to high potassium, kisspeptin, estradiol, and neurokinin B challenges; and injection of depolarizing current induced action potentials. Finally, we characterized developmental changes in transcriptomes of GnRH neurons using hESC, hiPSC, and mCh-hESC. The developmental pattern of transcriptomes was remarkably similar among the 3 cell lines. Collectively, human stem cell–derived GnRH neurons will be an important tool for establishing disease models to understand diseases, such as idiopathic hypothalamic hypogonadism, and testing contraceptive drugs.

The human testis can be infected by a large number of RNA and DNA viruses. While various RNA virus infections may induce orchitis and impair testicular functions, DNA virus infection rarely affects the testis. Mechanisms underlying the differential effects of RNA and DNA viral infections on the testis remain unclear. In the current study, we therefore examined the effects of viral RNA and DNA sensor signaling pathways on mouse Sertoli cells (SC) and Leydig cells (LC). The local injection of viral RNA analogue polyinosinic-polycytidylic acid [poly(I:C)] into the testis markedly disrupted spermatogenesis, whereas the injection of the herpes simplex virus (HSV) DNA analogue HSV60 did not affect spermatogenesis. Poly(I:C) dramatically induced the expression of the proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 in SC and LC through Toll-like receptor 3 and interferon β promoter stimulator 1 signaling pathways, impairing the integrity of the blood-testis barrier and testosterone synthesis. Poly(I:C)-induced TNF-α production thus plays a critical role in the impairment of cell functions. In contrast, HSV60 predominantly induced the expression of type 1 interferons and antiviral proteins via the DNA sensor signaling pathway, which did not affect testicular cell functions. Accordingly, the Zika virus induced high levels of TNF-α in SC and LC and impaired their respective cellular functions, whereas Herpes simplex virus type 2 principally induced antiviral responses and did not impair such functions. These results provide insights into the mechanisms by which RNA viral infections impair testicular functions.

The objective of this study was to determine the expression and functional role of a long noncoding RNA (lncRNA) MIAT (myocardial infarction–associated transcript) in leiomyoma pathogenesis. Leiomyoma compared with myometrium (n = 66) expressed significantly more MIAT that was independent of race/ethnicity and menstrual cycle phase but dependent on MED12 (mediator complex subunit 12) mutation status. Leiomyomas bearing the MED12 mutation expressed higher levels of MIAT and lower levels of microRNA 29 family (miR-29a, -b, and -c) compared with MED12 wild-type leiomyomas. Using luciferase reporter activity and RNA immunoprecipitation analysis, MIAT was shown to sponge the miR-29 family. In a 3-dimensional spheroid culture system, transient transfection of MIAT siRNA in leiomyoma smooth muscle cell (LSMC) spheroids resulted in upregulation of miR-29 family and downregulation of miR-29 targets, collagen type I (COL1A1), collagen type III (COL3A1), and TGF-β3 (transforming growth factor β-3). Treatment of LSMC spheroids with TGF-β3 induced COL1A1, COL3A1, and MIAT levels, but repressed miR-29 family expression. Knockdown of MIAT in LSMC spheroids blocked the effects of TGF-β3 on the induction of COL1A1 and COL3A1 expression. Collectively, these results underscore the physiological significance of MIAT in extracellular matrix accumulation in leiomyoma.

Defective placental implantation and vascularization with accompanying hypoxia contribute to preeclampsia (PE), a leading cause of maternal and neonatal morbidity and mortality. Genetic and epigenetic mechanisms underlying differentiation of proliferative cytotrophoblasts (CytTs) to multinucleated syncytiotrophoblast (SynT) are incompletely defined. The SynT performs key functions in nutrient and gas exchange, hormone production, and protection of the fetus from rejection by the maternal immune system. In this study, we used chromatin immunoprecipitation sequencing of midgestation human trophoblasts before CytT and after SynT differentiation in primary culture to analyze changes in binding of RNA polymerase II (Pol II) and of active and repressive histone marks during SynT differentiation. Our findings reveal that increased Pol II binding to promoters of a subset of genes during trophoblast differentiation was closely correlated with active histone marks. This gene set was enriched in those controlling immune response and immune modulation, including interferon-induced tetratricopeptide repeat and placenta-specific glycoprotein gene family members. By contrast, genes downregulated during SynT differentiation included proinflammatory transcription factors ERG1, cFOS, and cJUN, as well as members of the NR4A orphan nuclear receptor subfamily, NUR77, NURR1, and NOR1. Downregulation of proinflammatory transcription factors upon SynT differentiation was associated with decreased promoter enrichment of endogenous H3K27Ac and H3K9Ac and enhanced binding of H3K9me3 and histone deacetylase 1. However, promoter enrichment of H3K27me3 was low in both CytT and SynT and was not altered with changes in gene expression. These findings provide important insight into mechanisms underlying human trophoblast differentiation and may identify therapeutic targets for placental disorders, such as PE.

Tanycytes have recently been accepted as neural stem/progenitor cells in the postnatal hypothalamus. Persistent retina and anterior neural fold homeobox (Rax) expression is characteristic of tanycytes in contrast to its transient expression of whole hypothalamic precursors. In this study, we found that Rax^+ residual cells in the maturation phase of hypothalamic differentiation in mouse embryonic stem cell (mESC) cultures had similar characteristics to ventral tanycytes. They expressed typical neural stem/progenitor cell markers, including Sox2, vimentin, and nestin, and differentiated into mature neurons and glial cells. Quantitative RT-PCR analysis showed that Rax^+ residual cells expressed Fgf-10, Fgf-18, and Lhx2, which are expressed by ventral tanycytes. They highly expressed tanycyte-specific genes Dio2 and Gpr50 compared with Rax^+ early hypothalamic progenitor cells. Therefore, Rax^+ residual cells in the maturation phase of hypothalamic differentiation were considered to be more differentiated and similar to late progenitor cells and tanycytes. They self-renewed and formed neurospheres when cultured with exogenous FGF-2. Additionally, these Rax^+ neurospheres differentiated into three neuronal lineages (neurons, astrocytes, and oligodendrocytes), including neuropeptide Y^+ neuron, that are reported to be differentiated from ventral tanycytes toward the arcuate nuclei. Thus, Rax^+ residual cells were multipotent neural stem/progenitor cells. Rax^+ neurospheres were stably passaged and retained high Sox2 expression even after multiple passages. These results suggest the successful induction of Rax^+ tanycyte-like cells from mESCs [induced tanycyte-like (iTan) cells]. These hypothalamic neural stem/progenitor cells may have potential in regenerative medicine and as a research tool., ファイル公開日: 2020/07/01

Leptin has neurotrophic actions in the hippocampus to increase synapse formation and stimulate neuronal plasticity. Leptin also enhances cognition and has antidepressive and anxiolytic-like effects, two hippocampal-dependent behaviors. In contrast, mice lacking leptin or the long form of the leptin receptor (LepRb) have lower cortical volume and decreased memory and exhibit depressive-like behaviors. A number of the signaling pathways regulated by LepRb are known, but how membrane LepRb levels are regulated in the central nervous system is not well understood. Here, we show that the lysosomal inhibitor chloroquine increases LepRb expression in hippocampal cultures, suggesting that LepRb is degraded in the lysosome. Furthermore, we show that leptin increases surface expression of its own receptor by decreasing the level of ubiquitinated LepRbs. This decrease is mediated by the deubiquitinase ubiquitin-specific protease 8 (USP8), which we show is in complex with LepRb. Acute leptin stimulation increases USP8 activity. Moreover, leptin stimulates USP8 gene expression through cAMP response element–binding protein (CREB)–dependent transcription, an effect blocked by expression of a dominant-negative CREB or with short hairpin RNA knockdown of CREB. Increased expression of USP8 causes increased surface localization of LepRb, which in turn enhances leptin-mediated activation of the MAPK kinase/extracellular signal–regulated kinase pathway and CREB activation. Lastly, increased USP8 expression increases glutamatergic synapse formation in hippocampal cultures, an effect dependent on expression of LepRbs. Leptin-stimulated synapse formation also requires USP8. In conclusion, we show that USP8 deubiquitinates LepRb, thus inhibiting lysosomal degradation and enhancing surface localization of LepRb, which are essential for leptin-stimulated synaptogenesis in the hippocampus.

Stress decreases milk components such as milk protein and milk yield. The objective of this study was to investigate whether noradrenaline (NA) in milk constituted a factor associated with stress-induced changes in milk proteins such as β-casein. Breast milk obtained from eight healthy, nursing women contained NA at concentrations ranging from 12.7 to 115.5 nM. The expression of tyrosine hydroxylase (TH), a rate-limiting enzyme of NA synthesis, was observed in primary normal human mammary epithelial cells (HMECs), and in MCF-12A and MCF-10A cell lines. The mean NA concentration in culture medium used by MCF-12A transfected with TH small interfering RNA (siRNA) was significantly lower than that of cells transfected with control siRNA. NA concentration in milk in restraint-stressed nursing mice was significantly higher than that in nonstressed nursing mice, owing to elevated TH expression in the mammary epithelium. The mean β-casein concentration in milk in restraint-stressed mice was significantly lower than that in nonstressed mice. NA treatment resulted in a concentration-dependent decrease in β-casein expression in HMECs. β2 adrenergic receptor (ADRB2) expression was observed in HMECs, MCF-12A, and MCF-10A, and immunohistochemical analysis of ADRB2 using mammary epithelium sections obtained from mice at day 10 of lactation showed that ADRB2 was expressed at the apical membrane of mammary epithelium. Treatment with salbutamol, an ADRB2 stimulant, decreased β-casein expression in a concentration-dependent manner in MCF-12A. Our results showed that endogenous NA derived from mammary epithelial cells likely comprises one of the factors involved in stress-induced changes in milk proteins such as β-casein.

During spermatogenesis, microtubule (MT) cytoskeleton in Sertoli cells confers blood–testis barrier (BTB) function, but the regulators and mechanisms that modulate MT dynamics remain unexplored. In this study, we examined the role of calmodulin-regulated spectrin-associated protein (CAMSAP)2 (a member of the CAMSAP/Patronin protein family), and a minus-end targeting protein (−TIP) that binds to the minus-end (i.e., slow-growing end) of polarized MTs involved in determining MT length, in Sertoli cell function. CAMSAP2 was found to localize at discrete sites across the Sertoli cell cytosol, different from end-binding protein 1 (a microtubule plus-end tracking protein that binds to the plus-end of MTs), and colocalized with MTs. CAMSAP2 displayed a stage-specific expression pattern, appearing as tracklike structures across the seminiferous epithelium in adult rat testes that lay perpendicular to the basement membrane. CAMSAP2 knockdown by RNA interference was found to promote Sertoli cell tight junction (TJ) barrier function, illustrating its role in inducing TJ remodeling under physiological conditions. To further examine the regulatory role of CAMSAP2 in BTB dynamics, we used a perfluorooctanesulfonate (PFOS)-induced Sertoli cell injury model for investigations. CAMSAP2 knockdown blocked PFOS-induced Sertoli cell injury by promoting proper distribution of BTB-associated proteins at the cell–cell interface. This effect was mediated by the ability of CAMSAP2 knockdown to block PFOS-induced disruptive organization of MTs, but also F-actin, across cell cytosol through changes in cellular distribution/localization of MT- and actin-regulatory proteins. In summary, CAMSAP2 is a regulator of MT and actin dynamics in Sertoli cells to support BTB dynamics and spermatogenesis.

Fibroblast growth factor 23 (FGF23) secretion is facilitated by the PTH, particularly in hyperparathyroidism. The PTH also attenuates dentin matrix protein 1 (DMP1), which is produced by osteocytes to contribute to bone mineralization and suppress FGF23 expression. Nevertheless, it remains unknown whether attenuated DMP1 affects FGF23 expression in hyperparathyroidism. We examined their expression in bone tissue using a mouse model of primary hyperparathyroidism (PHPT). PHPT mice increased serum FGF23 levels, along with a high level of serum PTH. Fgf23 expression increased, and Dmp1 decreased significantly in the calvaria of PHPT mice compared with wild-type mice and primary osteoblasts treated with PTH. In UMR106 mature osteoblasts, PTH increased Fgf23 expression and decreased Dmp1 expression, and stimulation of protein kinase A (PKA) signaling by forskolin also increased Fgf23 expression and decreased Dmp1 expression in a dose-dependent manner, whereas inhibition of PKA signaling with 10-5 M H89 reversed the changes in Fgf23 and Dmp1 expression when cells were stimulated with PTH. Silencing Dmp1 along with PTH treatment led to an additive increase in Fgf23 expression, accompanied by additive phosphorylation of the cAMP-response element-binding protein. These results indicate that persistent and high levels of PTH lead to the continuous activation of PKA signaling in osteoblasts/osteocytes, resulting in an increase in FGF23 and a decrease in DMP1 in bone. Moreover, suppression of DMP1 enhanced FGF23 expression in PHPT, besides having a direct effect on PTH. These mechanisms may describe one of the pathogeneses behind the increase in FGF23 transcription in bone tissue in patients with PHPT.

Leptin has been proposed to modulate viscerosensory information directly at the level of vagal afferents. In support of this view, broad expression for the leptin receptor (Lepr) has previously been reported in vagal afferents. However, the exact identity and distribution of leptin-sensitive vagal afferents has not been elucidated. Using quantitative PCR, we found that the whole mouse nodose ganglion was predominantly enriched in the short form of Lepr, rather than its long form. Consistent with this observation, the acute administration of leptin did not stimulate JAK-STAT signaling in the nodose ganglion. Using chromogenic in situ hybridization in wild-type mice and several reporter mouse models, we demonstrated that Lepr mRNA was restricted to nonneuronal cells in the epineurium and parenchyma of the nodose ganglion and a subset of vagal afferents, which accounted for only 3% of all neuronal profiles. Double labeling studies further established that Lepr-expressing vagal afferents were Na(v)1.8-negative fibers that did not supply the peritoneal cavity. Finally, double chromogenic in situ hybridization revealed that many Lepr-expressing neurons coexpressed the angiotensin 1a receptor (At1ar), which is a gene expressed in baroreceptors. Taken together, our data challenge the commonly held view that Lepr is broadly expressed in vagal afferents. Instead, our data suggest that leptin may exert a previously unrecognized role, mainly via its short form, as a direct modulator of a very small group of At1ar-positive vagal fibers.

There is abundant evidence that low circulating testosterone levels in older men are associated with adverse cardiovascular outcomes; however, the direction of causality is unclear. Although there is burgeoning interest in the potential of androgen therapy in older men, the effect of androgens on cardiovascular regeneration in aging males remains poorly defined. We investigated the role of androgens in age-related impairment in ischemia-induced neovascularization. Castrated young (2 months) and old (24 months) male mice were subjected to unilateral hindlimb ischemia and treated with subdermal DHT or placebo Silastic implants. Blood flow recovery was enhanced by DHT treatment in young and old mice compared with age-matched placebo controls. DHT augmented angiogenesis in young mice and ameliorated age-related impairment in neovascularization in old mice. Administration of DHT was associated with increased hypoxia inducible factor-1α (HIF-1α) and stromal cell‒derived factor-1 expression in young mice, but not in old mice. In vitro, DHT-induced HIF-1α transcriptional activation was attenuated in fibroblasts from old mice. Interaction between androgen receptor (AR) and importins, key proteins that facilitate nuclear translocation of AR, was impaired with age. In contrast, DHT treatment stimulated the production and mobilization of Sca1+/CXCR4+ circulating progenitor cells in both young and old mice. DHT stimulated the migration and proangiogenic paracrine effect of ex vivo cultured bone marrow‒derived angiogenic cells from young and old mice. In conclusion, androgens ameliorated age-related impairment in ischemia-induced neovascularization. Although age-dependent dysfunction in androgen signaling attenuated some androgen effects on angiogenesis, provasculogenic effects of androgens were partially preserved with age.

Background During the transformation to dedifferentiated thyroid cancer (TC) types, the ability of papillary thyroid carcinomas (PTCs) to concentrate radioactive iodine might be lost, raising difficulty for the current therapy. circRNAs were proved to be implicated in the progression of various cancers. In this study, we aimed to investigate the functional role and mechanism of hsa_circ_0023990 in dedifferentiated TC. Methods The expression pattern of genes were detected using quantitative PCR or western blot assays. Cell proliferation was determined by CCK8, colony formation, EdU, and cell-cycle assays. Glycolysis was assessed using glucose uptake and lactate production assays. Luciferase reporter assay was performed to examine the interactions between miR-485-5p and hsa_circ_0023990 or FOXM1. Xenograft assay was allowed for observation of tumor growth in vivo. Results Hsa_circ_0023990 and FOXM1 were upregulated in dedifferentiated TC tissues and cell lines. The higher level of hsa_circ_0023900 could stimulate the proliferation and glycolysis of dedifferentiated TC cells via positively regulating FOXM1. Mechanistically, miR-485-5p was demonstrated to interact with hsa_circ_0023990 and FOXM1 and involved in the regulation of has_circ_0023990 and FOXM1 in TC biological processes. Conclusion Our results discovered the functional network of hsa_circ_0023990 in dedifferentiated TC development by facilitating cell proliferation and glycolysis via miR-485-5p/FOXM1 axis, implying that hsa_circ_0023990 might be a potential therapeutic target for the dedifferentiated TC treatment.

The CX3CL1-CX3CR1 system plays an important role in disease progression by regulating inflammation both positively and negatively. We reported previously that C-C chemokine receptors 2 and 5 promote obesity-associated adipose tissue inflammation and insulin resistance. Here, we demonstrate that CX3CL1-CX3CR1 signaling is involved in adipose tissue inflammation and insulin resistance in obese mice via adipose tissue macrophage recruitment and M1/M2 polarization. Cx3cl1 expression was persistently decreased in the epididymal white adipose tissue (eWAT) of high-fat diet-induced obese (DIO) mice, despite increased expression of other chemokines. Interestingly, in Cx3cr1−/− mice, glucose tolerance, insulin resistance, and hepatic steatosis induced by DIO or leptin deficiency were exacerbated. CX3CL1-CX3CR1 signaling deficiency resulted in reduced M2-polarized macrophage migration and an M1-dominant shift of macrophages within eWAT. Furthermore, transplantation of Cx3cr1−/− bone marrow was sufficient to impair glucose tolerance, insulin sensitivity, and regulation of M1/M2 status. Moreover, Cx3cl1 administration in vivo led to the attenuation of glucose intolerance and insulin resistance. Thus, therapy targeting the CX3CL1-CX3CR1 system may be beneficial in the treatment of type 2 diabetes by regulating M1/M2 macrophages.

The mammary gland is a hormone sensitive organ that is susceptible to endocrine-disrupting chemicals (EDCs) during the vulnerable periods of parous reorganization (ie, pregnancy, lactation, and involution). Pregnancy is believed to have long-term protective effects against breast cancer development; however, it is unknown if EDCs can alter this effect. We examined the long-term effects of propylparaben, a common preservative used in personal care products and foods, with estrogenic properties, on the parous mouse mammary gland. Pregnant BALB/c mice were treated with 0, 20, 100, or 10 000 µg/kg/day propylparaben throughout pregnancy and lactation. Unexposed nulliparous females were also evaluated. Five weeks post-involution, mammary glands were collected and assessed for changes in histomorphology, hormone receptor expression, immune cell number, and gene expression. For several parameters of mammary gland morphology, propylparaben reduced the effects of parity. Propylparaben also increased proliferation, but not stem cell number, and induced modest alterations to expression of ERα-mediated genes. Finally, propylparaben altered the effect of parity on the number of several immune cell types in the mammary gland. These results suggest that propylparaben, at levels relevant to human exposure, can interfere with the effects of parity on the mouse mammary gland and induce long-term alterations to mammary gland structure. Future studies should address if propylparaben exposures negate the protective effects of pregnancy on mammary cancer development.

Melatonin, a pineal gland hormone, has been suggested to treat postmenopausal osteoporosis due to its inhibitory effect on osteoclast differentiation. We previously reported that protein arginine methyltransferase 1 (PRMT1) was an important mediator of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. However, the relationship between melatonin and PRMT1 in osteoclast differentiation and estrogen deficiency–induced osteoporosis is unclear. In this study, we investigated the inhibitory mechanisms of melatonin in vitro and in vivo by focusing on PRMT1. Melatonin treatment effectively blocked RANKL-induced osteoclastogenesis by inhibiting PRMT1 and asymmetric dimethylarginine (ADMA) expression. RANKL-induced tumor necrosis factor receptor-associated factor 6 (TRAF6) and the phosphorylation of JNK were also suppressed by melatonin, and TRAF6 siRNA attenuated RANKL-induced p-JNK and PRMT1 production. Melatonin inhibited the transcriptional activity of NF-κB by interfering with the binding of PRMT1 and NF-κB subunit p65 in RANKL-treated bone marrow–derived macrophages. Our results also revealed that melatonin inhibits RANKL-induced PRMT1 expression through receptors-independent pathway. Thus, the anti-osteoclastogenic effect of melatonin was mediated by a cascade of inhibition of RANKL-induced TRAF6, JNK, PRMT1, and NF-κB signaling in melatonin receptors-independent pathway. In vivo, ovariectomy caused significant decreases in bone mineral density, but melatonin treatment alleviated the ovariectomized (OVX)-induced bone loss by inhibiting bone resorption. Furthermore, the expression PRMT1 and TRAP mRNA was upregulated in OVX-femurs, but effectively suppressed by melatonin injection. These findings suggest that melatonin inhibited osteoclast differentiation and estrogen deficiency–induced osteoporosis by suppressing RANKL-induced TRAF6, JNK, PRMT1, and NF-κB signaling cascades in melatonin receptors-independent pathway.

Context Healthy hyperplasic (many but smaller fat cells) white adipose tissue (WAT) expansion is mediated by recruitment, proliferation and/or differentiation of new fat cells. This process (adipogenesis) is controlled by transcriptional programs that have been mostly identified in rodents. Objective A systemic investigation of adipogenic human transcription factors (TFs) that are relevant for metabolic conditions has not been revealed previously. Methods TFs regulated in WAT by obesity, adipose morphology, cancer cachexia, and insulin resistance were selected from microarrays. Their role in differentiation of human adipose tissue-derived stem cells (hASC) was investigated by RNA interference (RNAi) screen. Lipid accumulation, cell number, and lipolysis were measured for all screened factors (148 TFs). RNA (RNAseq), protein (Western blot) expression, insulin, and catecholamine responsiveness were examined in hASC following siRNA treatment of selected target TFs. Results Analysis of TFs regulated by metabolic conditions in human WAT revealed that many of them belong to adipogenesis-regulating pathways. The RNAi screen identified 39 genes that affected fat cell differentiation in vitro, where 11 genes were novel. Of the latter JARID2 stood out as being necessary for formation of healthy fat cell metabolic phenotype by regulating expression of multiple fat cell phenotype-specific genes. Conclusion This comprehensive RNAi screening in hASC suggests that a large proportion of WAT TFs that are impacted by metabolic conditions might be important for hyperplastic adipose tissue expansion. The screen also identified JARID2 as a novel TF essential for the development of functional adipocytes.

Perinatal hypoxia severely disrupts cerebral metabolic and maturational programs beyond apoptotic cell death. Antiapoptotic treatments such as erythropoietin are suggested to improve outcomes in hypoxic brain injury; however, the results are controversial. We analyzed the neuroprotective effects of recombinant human growth hormone (rhGH) on regenerative mechanisms in the hypoxic developing mouse brain in comparison to controls. Using an established model of neonatal acute hypoxia (8% O2, 6 hours), P7 mice were treated intraperitoneally with rhGH (4000 µg/kg) 0, 12, and 24 hours after hypoxic exposure. After a regeneration period of 48 hours, expression of hypoxia-inducible neurotrophic factors (erythropoietin [EPO], vascular endothelial growth factor A [VEGF-A], insulin-like growth factors 1 and 2 [IGF-1/-2], IGF binding proteins) and proinflammatory markers was analyzed. In vitro experiments were performed using primary mouse cortical neurons (E14, DIV6). rhGH increased neuronal gene expression of EPO, IGF-1, and VEGF (P

Increased glucagon is a hallmark of diabetes and leads to worsening of the hyperglycemia, but the molecular mechanisms causing it are still unknown. We therefore investigated the possibility that microRNAs might be involved in the regulation of glucagon. Indeed, analysis of the glucagon 3′ untranslated region (UTR) revealed potential binding sites for miR-320a, and using luciferase reporter assays we found that miR-320a directly targets the 3′ UTRs of human and rodent glucagon. In addition, endogenous glucagon mRNA and protein expression as well as glucagon secretion were reduced in response to miR-320a overexpression, whereas inhibition of miR-320a upregulated glucagon expression. Interestingly, miR-320a expression was decreased by high glucose, and this was associated with an increase in glucagon expression in human islets and mouse αTC1-6 cells. Moreover, miR-320a overexpression completely blunted these effects. Importantly, miR-320a was also significantly downregulated in human islets of subjects with type 2 diabetes and this was accompanied by increased glucagon expression. Thus, our data suggest that glucose-induced downregulation of miR-320a may contribute to the paradoxical increase in glucagon observed in type 2 diabetes and reveal for the first time that glucagon expression is under the control by a microRNA providing novel insight into the abnormal regulation of glucagon in diabetes.

Exosomes are extracellular vesicles that can participate in autoimmune diseases. The purpose of this study was to explore whether circulating exosomes are involved in Graves’ disease (GD) pathogenesis. In this study, serum exosomes were extracted from 26 healthy controls (HC-EXO), 26 GD patients (GD-EXO), and 7 Graves’ ophthalmopathy patients (GO-EXO). For each group, the total protein content was detected, and thyrotropin receptor, insulin-like growth factor 1 receptor (IGF-1R), heat shock protein 60 (HSP60), and cluster of differentiation (CD) 63 expression were analyzed by Western blotting (WB). Healthy volunteer-derived peripheral blood mononuclear cells (PBMCs) and HC-EXO or GD-EXO were cocultured for 24 h, and immunofluorescence was used to observe the locations of the exosomes and toll-like receptor (TLR) 2/3. CD11c+TLR2+ and CD11c+TLR3+ cell percentages were determined by flow cytometry. Myeloid differentiation factor 88 (MyD88), toll/interleukin (IL)-1 receptor domain-containing adaptor inducing interferon-β (TRIF) and p-P65 expression were analyzed by WB. IL-6 and IL-1β supernatant levels were detected using enzyme-linked immunosorbent assay. The results showed that the total protein concentration was similar among GD-EXO, GO-EXO, and HC-EXO. IGF-1R and HSP60 expression was significantly higher in GD-EXO and GO-EXO than in HC-EXO. After coculturing PBMCs with GD-EXO or HC-EXO for 24 h, GD-EXO could bind to TLR2/3. GD-EXO significantly increased CD11c+TLR2+ and CD11c+TLR3+ cell percentages; MyD88, TRIF, and p-P65 protein expression; and IL-6 and IL-1β levels. In conclusion, we first demonstrated that GD-EXO and GO-EXO highly expressed IGF-1R and HSP60. GD-EXO may induce an inflammatory response through the TLR/NF-κB signaling pathway and be involved in the pathogenesis of GD.

Insulin secretion from pancreatic beta cells is tightly regulated by glucose and paracrine signals within the microenvironment of islets of Langerhans. Extracellular matrix from islet microcapillary endothelial cells (IMEC) affect beta-cell spreading and amplify insulin secretion. This study was aimed at investigating the hypothesis that contact-independent paracrine signals generated from IMEC may also modulate beta-cell insulin secretory functions. For this purpose, conditioned medium (CMp) preparations were prepared from primary cultures of rat IMEC and were used to simulate contact-independent beta cell–endothelial cell communication. Glucose-stimulated insulin secretion (GSIS) assays were then performed on freshly isolated rat islets and the INS-1E insulinoma cell line, followed by fractionation of the CMp, mass spectroscopic identification of the factor, and characterization of the mechanism of action. The IMEC-derived CMp markedly attenuated first- and second-phase GSIS in a time- and dose-dependent manner without altering cellular insulin content and cell viability. Size exclusion fractionation, chromatographic and mass-spectroscopic analyses of the CMp identified the attenuating factor as the enzyme triosephosphate isomerase (TPI). An antibody against TPI abrogated the attenuating activity of the CMp while recombinant human TPI (hTPI) attenuated GSIS from beta cells. This effect was reversed in the presence of tolbutamide in the GSIS assay. In silico docking simulation identified regions on the TPI dimer that were important for potential interactions with the extracellular epitopes of the sulfonylurea receptor in the complex. This study supports the hypothesis that an effective paracrine interaction exists between IMEC and beta cells and modulates glucose-induced insulin secretion via TPI–sulfonylurea receptor–KATP channel (SUR1-Kir6.2) complex attenuating interactions.

The molecular interactions between the maternal environment and the developing embryo are key for early pregnancy success and are influenced by factors such as maternal metabolic status. Our understanding of the mechanism(s) through which these individual nutritional stressors alter endometrial function and the in utero environment for early pregnancy success is, however, limited. Here we report, for the first time, the use of an endometrium-on-a-chip microfluidics approach to produce a multicellular endometrium in vitro. Isolated endometrial cells (epithelial and stromal) from the uteri of nonpregnant cows in the early luteal phase (Days 4-7) were seeded in the upper chamber of the device (epithelial cells; 4-6 × 104 cells/mL) and stromal cells seeded in the lower chamber (1.5-2 × 104 cells/mL). Exposure of cells to different concentrations of glucose (0.5, 5.0, or 50 mM) or insulin (Vehicle, 1 or 10 ng/mL) was performed at a flow rate of 1 µL/minute for 72 hours. Quantitative differences in the cellular transcriptome and the secreted proteome of in vitro–derived uterine luminal fluid were determined by RNA-sequencing and tandem mass tagging mass spectrometry, respectively. High glucose concentrations altered 21 and 191 protein-coding genes in epithelial and stromal cells, respectively (P

A correct biosynthetic activity is thought to be essential for the long-term function and survival of islet cells in culture and possibly also after islet transplantation. Compared to the secretory activity, biosynthetic activity has been poorly studied in pancreatic islet cells. Here we aimed to assess biosynthetic activity at the single cell level to investigate if protein synthesis is dependent on secretagogues and increased as a consequence of hormonal secretion. Biosynthetic activity in rat islet cells was studied at the single cell level using O-propargyl-puromycin (OPP) that incorporates into newly translated proteins and chemically ligates to a fluorescent dye by “click” reaction. Heterogeneous biosynthetic activity was observed between the four islet cell types, with delta cells showing the higher relative protein biosynthesis. Beta cells protein biosynthesis was increased in response to glucose while 3-isobutyl-1-methylxanthine and phorbol-12-myristate-13-acetate, 2 drugs known to stimulate insulin secretion, had no similar effect on protein biosynthesis. However, after several hours of secretion, protein biosynthesis remained high even when cells were challenged to basal conditions. These results suggest that mechanisms regulating secretion and biosynthesis in islet cells are different, with glucose directly triggering beta cells protein biosynthesis, independently of insulin secretion. Furthermore, this OPP labeling approach is a promising method to identify newly synthesized proteins under various physiological and pathological conditions.

Prolactin (PRL), a hormone involved in lactation, is mainly produced and secreted by the lactotrophs of the anterior pituitary (AP) gland. We previously reported a method to generate functional adrenocorticotropic hormone-producing cells by differentiating the AP and hypothalamus simultaneously from human induced pluripotent stem cells (iPSCs). However, PRL-producing cells in the induced AP have not been investigated. Here, we confirmed the presence of PRL-producing cells and evaluated their endocrine functions. We differentiated pituitary cells from human iPSCs using serum-free floating culture of embryoid-like aggregates with quick reaggregation (SFEB-q) method and evaluated the appearance and function of PRL-producing cells. Secretion of PRL from the differentiated aggregates was confirmed, which increased with further culture. Fluorescence immunostaining and immunoelectron microscopy revealed PRL-producing cells and PRL-positive secretory granules, respectively. PRL secretion was promoted by various prolactin secretagogues such as thyrotropin-releasing hormone, vasoactive intestinal peptide, and prolactin-releasing peptide, and inhibited by bromocriptine. Moreover, the presence of tyrosine hydroxylase-positive dopaminergic nerves in the hypothalamic tissue area around the center of the aggregates connecting to PRL-producing cells indicated the possibility of recapitulating PRL regulatory mechanisms through the hypothalamus. In conclusion, we generated pituitary lactotrophs from human iPSCs; these displayed similar secretory responsiveness as human pituitary cells in vivo. In the future, this is expected to be used as a model of human PRL-producing cells for various studies, such as drug discovery, prediction of side effects, and elucidation of tumorigenic mechanisms using disease-specific iPSCs. Furthermore, it may help to develop regenerative medicine for the pituitary gland.

Adrenomedullin (AM) is a peptide hormone with multiple physiological functions, which are regulated by its receptor activity–modifying proteins, RAMP2 and RAMP3. We previously reported that AM or RAMP2 knockout (KO) (AM–/–, RAMP2–/–) is embryonically lethal in mice, whereas RAMP3–/– mice are apparently normal. AM, RAMP2, and RAMP3 are all highly expressed in the heart; however, their functions there are not fully understood. Here, we analyzed the pathophysiological functions of the AM-RAMP2 and AM-RAMP3 systems in hearts subjected to cardiovascular stress. Cardiomyocyte-specific RAMP2–/– (C-RAMP2–/–) and RAMP3–/– showed no apparent heart failure at base line. After 1 week of transverse aortic constriction (TAC), however, C-RAMP2–/– exhibited significant cardiac hypertrophy, decreased ejection fraction, and increased fibrosis compared with wild-type mice. Both dP/dtmax and dP/dtmin were significantly reduced in C-RAMP2–/–, indicating reduced ventricular contractility and relaxation. Exposing C-RAMP2–/– cardiomyocytes to isoproterenol enhanced their hypertrophy and oxidative stress compared with wild-type cells. C-RAMP2–/– cardiomyocytes also contained fewer viable mitochondria and showed reduced mitochondrial membrane potential and respiratory capacity. RAMP3–/– also showed reduced systolic function and enhanced fibrosis after TAC, but those only became apparent after 4 weeks. A reduction in cardiac lymphatic vessels was the characteristic feature in RAMP3–/–. These observations indicate the AM-RAMP2 system is necessary for early adaptation to cardiovascular stress through regulation of cardiac mitochondria. AM-RAMP3 is necessary for later adaptation through regulation of lymphatic vessels. The AM-RAMP2 and AM-RAMP3 systems thus play separate critical roles in the maintenance of cardiovascular homeostasis against cardiovascular stress.

Uterine leiomyoma (LM) is the most common tumor in women and can cause severe morbidity. Leiomyoma growth requires the maintenance and proliferation of a stem cell population. Dysregulated deoxyribonucleic acid (DNA) methylation has been reported in LM, but its role in LM stem cell regulation remains unclear. Here, we fluorescence-activated cell sorting (FACS)-sorted cells from human LM tissues into 3 populations: LM stem cell-like cells (LSC, 5%), LM intermediate cells (LIC, 7%), and differentiated LM cells (LDC, 88%), and we analyzed the transcriptome and epigenetic landscape of LM cells at different differentiation stages. Leiomyoma stem cell-like cells harbored a unique methylome, with 8862 differentially methylated regions compared to LIC and 9444 compared to LDC, most of which were hypermethylated. Consistent with global hypermethylation, transcript levels of TET1 and TET3 methylcytosine dioxygenases were lower in LSC. Integrative analyses revealed an inverse relationship between methylation and gene expression changes during LSC differentiation. In LSC, hypermethylation suppressed the genes important for myometrium- and LM-associated functions, including muscle contraction and hormone action, to maintain stemness. The hypomethylating drug, 5'-Aza, stimulated LSC differentiation, depleting the stem cell population and inhibiting tumor initiation. Our data suggest that DNA methylation maintains the pool of LSC, which is critical for the regeneration of LM tumors.

The free fatty acid receptor G protein-coupled receptor 120 (GPR120) is expressed in pancreatic islets, but its specific cell distribution and function have not been fully established. In this study, a GPR120-IRES-EGFP knockin (KI) mouse was generated to identify GPR120-expressing cells with enhanced green fluorescence proteins (EGFP). EGFP-positive cells collected from KI mouse islets by flow cytometry had a significantly higher expression of pancreatic polypeptide (PP) evidenced by reverse transcriptase (RT)-quantitative polymerase chain reaction (qPCR). Single-cell RT-PCR and immunocytochemical double staining also demonstrated the coexpression of GPR120 with PP in mouse islets. The GPR120-specific agonist TUG-891 significantly increased plasma PP levels in mice. TUG-891 significantly increased PP levels in islet medium in vitro, which was markedly attenuated by GPR120 small interfering RNA treatment. TUG-891–stimulated PP secretion in islets was fully blocked by pretreatment with YM-254890 (a Gq protein inhibitor), U73122 (a phospholipase C inhibitor), or thapsigargin (an inducer of endoplasmic reticulum Ca2+ depletion), respectively. TUG-891 triggered the increase in intracellular free Ca2+ concentrations ([Ca2+]i) in PP cells, which was also eliminated by YM-254890, U73122, or thapsigargin. GPR120 gene expression was significantly reduced in islets of high-fat diet (HFD)-induced obese mice. TUG-891–stimulated PP secretion was also significantly diminished in vivo and in vitro in HFD-induced obese mice compared with that in normal-chow diet control mice. In summary, this study demonstrated that GPR120 is expressed in mouse islet PP cells and GPR120 activation stimulated PP secretion via the Gq/PLC-Ca2+ signaling pathway in normal-chow diet mice but with diminished effects in HFD-induced obese mice.

The biologically active estrogen estradiol has important roles in adult brain physiology and sexual behavior. A single gene, Cyp19a1, encodes aromatase, the enzyme that catalyzes the conversion of testosterone to estradiol in the testis and brain of male mice. Estradiol formation was shown to regulate sexual activity in various species, but the relative contributions to sexual behavior of estrogen that arises in the brain versus from the gonads remained unclear. To determine the role of brain aromatase in regulating male sexual activity, we generated a brain-specific aromatase knockout (bArKO) mouse. A newly generated whole-body total aromatase knockout mouse of the same genetic background served as a positive control. Here we demonstrate that local aromatase expression and estrogen production in the brain is partially required for male sexual behavior and sex hormone homeostasis. Male bArKO mice exhibited decreased sexual activity in the presence of strikingly elevated circulating testosterone. In castrated adult bArKO mice, administration of testosterone only partially restored sexual behavior; full sexual behavior, however, was achieved only when both estradiol and testosterone were administered together. Thus, aromatase in the brain is, in part, necessary for testosterone-dependent male sexual activity. We also found that brain aromatase is required for negative feedback regulation of circulating testosterone of testicular origin. Our findings suggest testosterone activates male sexual behavior in part via conversion to estradiol in the brain. These studies provide foundational evidence that sexual behavior may be modified through inhibition or enhancement of brain aromatase enzyme activity and/or utilization of selective estrogen receptor modulators.

Differentiation of pluripotent stem cells into functional parathyroid-like cells would accelerate development of important therapeutic options for subjects with parathyroid-related disorders, from the design and screening of novel pharmaceutical agents to the development of durable cellular therapies. We have established a highly reproducible directed differentiation approach leading to PTH-expressing cells from human embryonic stem cells and induced pluripotent stem cells. We accomplished this through the comparison of multiple different basal media, the inclusion of the CDK inhibitor PD0332991 in both definitive endoderm and anterior foregut endoderm stages, and a 2-stage pharyngeal endoderm series. This is the first protocol to reproducibly establish PTH-expressing cells from human pluripotent stem cells and represents a first step toward the development of functional parathyroid cells with broad applicability for medicinal and scientific investigation.

Mesenchymal stem cells can differentiate into mature chondrocytes, osteoblasts, and adipocytes. Excessive and dysfunctional visceral adipocytes increase upon menopause and importantly contribute to altered metabolism in postmenopausal women. We previously showed both plasma membrane and nuclear estrogen receptors alpha (ERα) with endogenous estrogen are required to suppress adipogenesis in vivo. Here we determined mechanisms by which these liganded ER pools collaborate to inhibit the peroxisome proliferator-activated gamma (PPARγ) gene and subsequent progenitor differentiation. In 3T3-L1 pre-adipocytes and adipose-derived stem cells (ADSC), membrane ERα signaled through phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) to enhance ERα nuclear localization, importantly at the PPARγ gene promoter. AKT also increased overall abundance and recruitment of co-repressors GATA3, β-catenin, and TCF4 to the PPARγ promoter. Membrane ERα signaling additionally enhanced wingless-integrated (Wnt)1 and 10b expression. The components of the repressor complex were required for estrogen to inhibit rosiglitazone-induced differentiation of ADSC and 3T3-L1 cells to mature adipocytes. These mechanisms whereby ER cellular pools collaborate to inhibit gene expression limit progenitor differentiation to mature adipocytes.

The adipokine leptin regulates energy homeostasis through ubiquitously expressed leptin receptors. Leptin has a number of major signaling targets in the brain, including cells of the anterior pituitary (AP). We have previously reported that mice lacking leptin receptors in AP somatotropes display growth hormone (GH) deficiency, metabolic dysfunction, and adult-onset obesity. Among other targets, leptin signaling promotes increased levels of the pituitary transcription factor POU1F1, which in turn regulates the specification of somatotrope, lactotrope, and thyrotrope cell lineages within the AP. Leptin’s mechanism of action on somatotropes is sex dependent, with females demonstrating posttranscriptional control of Pou1f1 messenger RNA (mRNA) translation. Here, we report that the stem cell marker and mRNA translational control protein, Musashi1, exerts repression of the Pou1f1 mRNA. In female somatotropes, Msi1 mRNA and protein levels are increased in the mouse model that lacks leptin signaling (Gh-CRE Lepr-null), coincident with lack of POU1f1 protein, despite normal levels of Pou1f1 mRNA. Single-cell RNA sequencing of pituitary cells from control female animals indicates that both Msi1 and Pou1f1 mRNAs are expressed in Gh-expressing somatotropes, and immunocytochemistry confirms that Musashi1 protein is present in the somatotrope cell population. We demonstrate that Musashi interacts directly with the Pou1f1 mRNA 3′ untranslated region and exerts translational repression of a Pou1f1 mRNA translation reporter in a leptin-sensitive manner. Musashi immunoprecipitation from whole pituitary reveals coassociated Pou1f1 mRNA. These findings suggest a mechanism in which leptin stimulation is required to reverse Musashi-mediated Pou1f1 mRNA translational control to coordinate AP somatotrope function with metabolic status.

Restoration of spermatogenesis and fertility is a major issue to be solved in male mammals with hypogonadotropic hypogonadism. Kiss1 knockout (KO) male mice are postulated to be a suitable animal model to investigate if hormonal replacement rescues spermatogenesis in mammals with this severe reproductive hormone deficiency, because KO mice replicate the hypothalamic disorder causing hypogonadism. The present study investigated whether testosterone supplementation was able to restore spermatogenesis and in vitro fertilization ability in Kiss1 KO mice. To this end, spermatogenesis, in vitro fertilization ability of Kiss1 KO sperm, and preimplantation development of wild-type embryos inseminated with Kiss1 KO sperm, were examined. The newly generated Kiss1 KO male mice showed infertility with cryptorchidism. Subcutaneous testosterone supplementation for 6 weeks restored plasma and intratesticular testosterone levels, elicited testicular descent, and induced complete spermatogenesis from spermatocytes to elongated spermatids in the testis, resulting in an increase in epididymal sperm number in testosterone-supplemented Kiss1 KO male mice. Epididymal sperm derived from the testosterone-supplemented Kiss1 KO mice showed normal in vitro fertilization ability, and the fertilized eggs showed normal preimplantation development, while the males failed to impregnate females. These results suggest that the failure of spermatogenesis in Kiss1 KO mice is mainly due to a lack of testosterone production, and that Kiss1 KO sperm are capable of fertilizing eggs if the animals receive the appropriate testosterone supplementation without local kisspeptin signaling in the testis and epididymis. Thus, testosterone supplementation would restore spermatogenesis of male mammals showing hypogonadotropic hypogonadism with genetic inactivation of the KISS1/Kiss1 gene.

Mitochondria play important roles in ovarian follicle development. Mitochondrial dysfunction, including mitochondrial gene deficiency, impairs ovarian development. Here, we explored the role and mechanism of mitochondrial inner membrane gene Immp2l in ovarian follicle growth and development. Our results revealed that female Immp2l-/- mice were infertile, whereas Immp2l+/- mice were normal. Body and ovarian weights were reduced in the female Immp2l-/- mice, ovarian follicle growth and development were stunted in the secondary follicle stage. Although a few ovarian follicles were ovulated, the oocytes were not fertilized because of mitochondrial dysfunction. Increased oxidative stress, decreased estrogen levels, and altered genes expression of Wnt/β-catenin and steroid hormone synthesis pathways were observed in 28-day-old Immp2l-/- mice. The Immp2l mutation accelerated ovarian aging process, as no ovarian follicles were detected by age 5 months in Immp2l-/- mice. All the aforementioned changes in the Immp2l-/- mice were reversed by administration of antioxidant melatonin to the Immp2l-/- mice. Furthermore, our in vitro study using Immp2l knockdown granulosa cells confirmed that the Immp2l downregulation induced granulosa cell aging by enhancing reactive oxygen species (ROS) levels, suppressing Wnt16, increasing β-catenin, and decreasing steroid hormone synthesis gene cyp19a1 and estrogen levels, accompanied by an increase in the aging phenotype of granulosa cells. Melatonin treatment delayed granulosa cell aging progression. Taken together, Immp2l causes ovarian aging through the ROS-Wnt/β-catenin-estrogen (cyp19a1) pathway, which can be reversed by melatonin treatment.