Notch signaling is significantly suppressed in basal cell carcinomas and activation induces basal cell carcinoma cell apoptosis.

A subset of basal cell carcinomas (BCCs) are directly derived from hair follicles (HFs). In some respects, HFs can be defined as 'ordered' skin appendage growths, while BCCs can be regarded as 'disordered' skin appendage growths. The aim of the present study was to examine HFs and BCCs to define the expression of common and unique signaling pathways in each skin appendage. Human nodular BCCs, along with HFs and non­follicular skin epithelium from normal individuals, were examined using microarrays, qPCR, and immunohistochemistry. Subsequently, BCC cells and root sheath keratinocyte cells from HFs were cultured and treated with Notch signaling peptide Jagged1 (JAG1). Gene expression, protein levels, and cell apoptosis susceptibility were assessed using qPCR, immunoblotting, and flow cytometry, respectively. Specific molecular mechanisms were found to be involved in the process of cell self­renewal in the HFs and BCCs, including Notch and Hedgehog signaling pathways. However, several key Notch signaling factors showed significant differential expression in BCCs compared with HFs. Stimulating Notch signaling with JAG1 induced apoptosis of BCC cells by increasing Fas ligand expression and downstream caspase-8 activation. The present study showed that Notch signaling pathway activity is suppressed in BCCs, and is highly expressed in HFs. Elements of the Notch pathway could, therefore, represent targets for the treatment of BCCs and potentially in hair follicle engineering.

Low Thyroid Hormone in Early Pregnancy Is Associated With an Increased Risk of Gestational Diabetes Mellitus.

CONTEXT: Although thyroid dysfunction in early pregnancy may have adverse effects on pregnancy outcome and offspring, few prospective studies have evaluated these effects. OBJECTIVE: Our aim was to evaluate the correlations between different thyroid hormone levels in early pregnancy and the incidence of gestational diabetes mellitus (GDM). SETTING AND PARTICIPANTS: The study comprised 27 513 mothers who provided early pregnancy serum samples for analyses of thyroid function. GDM was diagnosed using a 2 hours, 75-g oral glucose tolerance test, and the mothers were grouped and compared according to the results. MAIN OUTCOME MEASURES: We focused on GDM during the index pregnancy. RESULTS: The incidence of GDM in pregnant women tended to increase with age (5.83%, 10.18%, 14.95%, and 22.40%; P < .0001). The incidence of GDM increased with increasing prepregnancy body mass index (P < .0001). Pregnant women with a family history of diabetes had a much higher incidence of GDM than those without a family history of diabetes (21.09% vs 12.92%; P < .0001). The level of free T4 (FT4) in early pregnancy in GDM women was lower than that in non GDM women (P < .0001). With increasing early pregnancy FT4, the rate of incident GDM was decreasing (P < .0001). CONCLUSIONS: Low thyroid hormone levels in early pregnancy are a risk factor for GDM incidence.

Identification of Autoantigen Epitopes in Alopecia Areata.

Alopecia areata (AA) is believed to be a cell-mediated autoimmune hair loss disease. Both CD4 and cytotoxic CD8 T cells (CTLs) are important for the onset and progression of AA. Hair follicle (HF) keratinocyte and/or melanocyte antigen epitopes are suspected potential targets of autoreactive CTLs, but the specific epitopes have not yet been identified. We investigated the potential for a panel of known epitopes, expressed by HF keratinocytes and melanocytes, to induce activation of CTL populations in peripheral blood mononuclear cells. Specific synthetic epitopes derived from HF antigens trichohyalin and tyrosinase-related protein-2 induced significantly higher frequencies of response in AA CTLs compared with healthy controls (IFN-gamma secretion). Apoptosis assays revealed conditioned media from AA peripheral blood mononuclear cells stimulated with trichohyalin peptides elevated the expression of apoptosis markers in primary HF keratinocytes. A cytokine array revealed higher expression of IL-13 and chemokine ligand 5 (CCL5, RANTES) from AA peripheral blood mononuclear cells stimulated with trichohyalin peptides compared with controls. The data indicate that AA affected subjects present with an increased frequency of CTLs responsive to epitopes originating from keratinocytes and melanocytes; the activated CTLs secreted soluble factors that induced apoptosis in HF keratinocytes. Potentially, CTL response to self-antigen epitopes, particularly trichohyalin epitopes, could be a prognostic marker for human AA.

Comparison of the Reference Intervals Used for the Evaluation of Maternal Thyroid Function During Pregnancy Using Sequential and Nonsequential Methods.

BACKGROUND: Maternal thyroid dysfunction is common during pregnancy, and physiological changes during pregnancy can lead to the overdiagnosis of hyperthyroidism and misdiagnosis of hypothyroidism with nongestation-specific reference intervals. Our aim was to compare sequential with nonsequential methods for the evaluation of thyroid function in pregnant women. METHODS: We tested pregnant women who underwent their trimester prenatal screening at our hospital from February 2011 to September 2012 for serum thyroid stimulating hormone (TSH) and free thyroxine (FT4) using the Abbott and Roche kits. There were 447 and 200 patients enrolled in the nonsequential and sequential groups, respectively. The central 95% range between the 2.5th and the 97.5th percentiles was used as the reference interval for the thyroid function parameter. RESULTS: The nonsequential group exhibited a significantly larger degree of dispersion in the TSH reference interval during the 2nd and 3rd trimesters as measured using both the Abbott and Roche kits (all P < 0.05). The TSH reference intervals were significantly larger in the nonsequential group than in the sequential group during the 3rd trimester as measured with both the Abbott (4.95 vs. 3.77 mU/L, P < 0.001) and Roche kits (6.62 vs. 5.01 mU/L, P = 0.004). The nonsequential group had a significantly larger FT4 reference interval as measured with the Abbott kit during all trimesters (12.64 vs. 5.82 pmol/L; 7.96 vs. 4.77 pmol/L; 8.10 vs. 4.77 pmol/L, respectively, all P < 0.05), whereas a significantly larger FT4 reference interval was only observed during the 2nd trimester with the Roche kit (7.76 vs. 5.52 pmol/L, P = 0.002). CONCLUSIONS: It was more reasonable to establish reference intervals for the evaluation of maternal thyroid function using the sequential method during each trimester of pregnancy. Moreover, the exclusion of pregnancy-related complications should be considered in the inclusion criteria for thyroid function tests.

Activin A, B and AB decrease progesterone production by down-regulating StAR in human granulosa cells.

Activins are homo- or heterodimers of inhibin ß subunits that play important roles in the reproductive system. Our previous work has shown that activins A (ßAßA), B (ßBßB) and AB (ßAßB) induce aromatase/estradiol, but suppress StAR/progesterone production in human granulosa-lutein cells. However, the underlying molecular determinants of these effects have not been examined. In this continuing study, we used immortalized human granulosa cells (SVOG) to investigate the effects of activins in regulating StAR/progesterone and the potential mechanisms of action. In SVOG cells, activins A, B and AB produced comparable down-regulation of StAR expression and progesterone production. In addition, all three activin isoforms induced equivalent phosphorylation of both SMAD2 and SMAD3. Importantly, the activin-induced down-regulation of StAR, increase in SMAD2/3 phosphorylation, and decrease in progesterone were abolished by the TGF-ß type I receptor inhibitor SB431542. Interestingly, the small interfering RNA-mediated knockdown of ALK4 but not ALK5 reversed the activin-induced suppression of StAR. Furthermore, the knockdown of SMAD4 or SMAD2 but not SMAD3 abolished the inhibitory effects of all three activin isoforms on StAR expression. These results provide evidence that activins A, B and AB down-regulate StAR expression and decrease progesterone production in human granulosa cells, likely via an ALK4-mediated SMAD2/SMAD4-dependent pathway. Our findings provide important insights into the molecular mechanisms underlying the regulatory effects of activins on human granulosa cell steroidogenesis.

High Maternal Serum Estradiol Levels Induce Dyslipidemia in Human Newborns via a Hepatic HMGCR Estrogen Response Element.

UNLABELLED: While the intrauterine environment is essential for the health of offspring, the impact of high maternal serum estradiol (E2) on lipid metabolism in offspring and the mechanisms are unknown. We found that ovarian stimulation (OS) could result in high E2 levels in women throughout pregnancy. Strikingly, their newborns showed elevated total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels that were positively related with E2 in newborns. In vitro, E2 dose-dependently stimulated TC and LDL-C secretion, and increased expression of the cholesterol synthesis rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) in HepG2 cells and mouse fetal hepatocytes. In vivo, high maternal E2 was detected and fetal livers also showed significantly higher HMGCR expression in an OS mouse model. Notably, an estrogen response element (ERE) was identified in the HMGCR promoter, indicating that high maternal serum E2 could up-regulate HMGCR expression in fetal hepatocytes via an ERE that in turn induces elevated levels of TC and LDL-C in offspring. CONCLUSION: OS can induce a high maternal E2 environment, which up-regulates HMGCR expression in fetal hepatocytes via an ERE in the promoter, and induces elevated levels of TC and LDL-C in newborns that may be related to increased risk of metabolic disease in adulthood.

Actl6a protects embryonic stem cells from differentiating into primitive endoderm.

Actl6a (actin-like protein 6A, also known as Baf53a or Arp4) is a subunit shared by multiple complexes including esBAF, INO80, and Tip60-p400, whose main components (Brg1, Ino80, and p400, respectively) are crucial for the maintenance of embryonic stem cells (ESCs). However, whether and how Actl6a functions in ESCs has not been investigated. ESCs originate from the epiblast (EPI) that is derived from the inner cell mass (ICM) in blastocysts, which also give rise to primitive endoderm (PrE). The molecular mechanisms for EPI/PrE specification remain unclear. In this study, we provide the first evidence that Actl6a can protect mouse ESCs (mESCs) from differentiating into PrE. While RNAi knockdown of Actl6a, which appeared highly expressed in mESCs and downregulated during differentiation, induced mESCs to differentiate towards the PrE lineage, ectopic expression of Actl6a was able to repress PrE differentiation. Our work also revealed that Actl6a could interact with Nanog and Sox2 and promote Nanog binding to pluripotency genes such as Oct4 and Sox2. Interestingly, cells depleted of p400, but not of Brg1 or Ino80, displayed similar PrE differentiation patterns. Mutant Actl6a with impaired ability to bind Tip60 and p400 failed to block PrE differentiation induced by Actl6a dysfunction. Finally, we showed that Actl6a could target to the promoters of key PrE regulators (e.g., Sall4 and Fgf4), repressing their expression and inhibiting PrE differentiation. Our findings uncover a novel function of Actl6a in mESCs, where it acts as a gatekeeper to prevent mESCs from entering into the PrE lineage through a Yin/Yang regulating pattern.

Ten-eleven translocation 1 (Tet1) is regulated by O-linked N-acetylglucosamine transferase (Ogt) for target gene repression in mouse embryonic stem cells.

As a member of the Tet (Ten-eleven translocation) family proteins that can convert 5-methylcytosine (5mC) to 5-hydroxylmethylcytosine (5hmC), Tet1 has been implicated in regulating global DNA demethylation and gene expression. Tet1 is highly expressed in embryonic stem (ES) cells and appears primarily to repress developmental genes for maintaining pluripotency. To understand how Tet1 may regulate gene expression, we conducted large scale immunoprecipitation followed by mass spectrometry of endogenous Tet1 in mouse ES cells. We found that Tet1 could interact with multiple chromatin regulators, including Sin3A and NuRD complexes. In addition, we showed that Tet1 could also interact with the O-GlcNAc transferase (Ogt) and be O-GlcNAcylated. Depletion of Ogt led to reduced Tet1 and 5hmC levels on Tet1-target genes, whereas ectopic expression of wild-type but not enzymatically inactive Ogt increased Tet1 levels. Mutation of the putative O-GlcNAcylation site on Tet1 led to decreased O-GlcNAcylation and level of the Tet1 protein. Our results suggest that O-GlcNAcylation can positively regulate Tet1 protein concentration and indicate that Tet1-mediated 5hmC modification and target repression is controlled by Ogt.

Growth differentiation factor 9 (GDF9) suppresses follistatin and follistatin-like 3 production in human granulosa-lutein cells.

BACKGROUND: We have demonstrated that growth differentiation factor 9 (GDF9) enhances activin A-induced inhibin ß(B)-subunit mRNA levels in human granulosa-lutein (hGL) cells by regulating receptors and key intracellular components of the activin signaling pathway. However, we could not exclude its effects on follistatin (FST) and follistatin-like 3 (FSTL3), well recognized extracellular inhibitors of activin A. METHODOLOGY: hGL cells from women undergoing in vitro fertilization (IVF) treatment were cultured with and without siRNA transfection of FST, FSTL3 or GDF9 and then treated with GDF9, activin A, FST, FSTL3 or combinations. FST, FSTL3 and inhibin ß(B)-subunit mRNA, and FST, FSTL3 and inhibin B protein levels were assessed with real-time RT-PCR and ELISA, respectively. Data were log transformed before ANOVA followed by Tukey's test. PRINCIPAL FINDINGS: GDF9 suppressed basal FST and FSTL3 mRNA and protein levels in a time- and dose-dependent manner and inhibited activin A-induced FST and FSTL3 mRNA and protein expression, effects attenuated by BMPR2 extracellular domain (BMPR2 ECD), a GDF9 antagonist. After GDF9 siRNA transfection, basal and activin A-induced FST and FSTL3 mRNA and protein levels increased, but changes were reversed by adding GDF9. Reduced endogenous FST or FSTL3 expression with corresponding siRNA transfection augmented activin A-induced inhibin ß(B)-subunit mRNA levels as well as inhibin B levels (P values all <0.05). Furthermore, the enhancing effects of GDF9 in activin A-induced inhibin ß(B)-subunit mRNA and inhibin B production were attenuated by adding FST. CONCLUSION: GDF9 decreases basal and activin A-induced FST and FSTL3 expression, and this explains, in part, its enhancing effects on activin A-induced inhibin ß(B)-subunit mRNA expression and inhibin B production in hGL cells.

Growth differentiation factor 9 reverses activin A suppression of steroidogenic acute regulatory protein expression and progesterone production in human granulosa-lutein cells.

BACKGROUND: We have reported that growth differentiation factor 9 (GDF9) can enhance activin A (ß(A)ß(A))-induced inhibin B (αß(B)) secretion in human granulosa-lutein (hGL) cells, but its effects on steroidogenic acute regulatory protein (StAR), ovarian steroidogenic enzymes, and progesterone production are unknown. We undertook this study to further evaluate GDF9 in this regard. METHODS: hGL cells from women undergoing in vitro fertilization treatment were cultured with and without small interfering RNA (siRNA) transfection targeted at inhibin α-subunit or GDF9 before treatment with GDF9, activin A, FSH, or combinations. We compared StAR, P450 side-chain cleavage enzyme, and 3ß-hydroxysteroid dehydrogenase expression in hGL cells and progesterone levels in culture media after these treatments. mRNA, protein, and hormone levels were assessed with real-time RT-PCR, immunoblotting, and ELISA, respectively. Data were analyzed by ANOVA followed by Tukey's test. RESULTS: Activin A alone reduced basal and FSH-induced progesterone production by decreasing the expression of StAR protein, which regulates the rate-limiting step in steroidogenesis but not P450 side-chain cleavage enzyme and 3ß-hydroxysteroid dehydrogenase. GDF9 attenuated these activin A effects on StAR and progesterone. After transfection of α-subunit siRNA, activin A level increased (P < 0.001), whereas basal and activin A-induced inhibin B levels (with and without GDF9) decreased. Furthermore, the effects of GDF9 in reversing activin A suppression of progesterone production were attenuated (P < 0.001). Transfection of GDF9 siRNA decreased GDF9 as expected and led to lower StAR expression and progesterone secretion than those observed with activin A treatment alone. CONCLUSION: GDF9 attenuates the suppressive effects of activin A on StAR expression and progesterone production by increasing the expression of inhibin B, which acts as an activin A competitor.

Effects of endogenous growth differentiation factor 9 on activin A-induced inhibin B production in human granulosa-lutein cells.

BACKGROUND: We recently reported on the effects of exogenous growth differentiation factor 9 (GDF9) in enhancing activin A-induced inhibin beta(B)-subunit mRNA and inhibin B levels in human granulosa-lutein (hGL) cells by modulating key components of the activin signaling pathway. We undertook the following study to characterize the role of endogenous GDF9 in this regard. METHODS: We compared inhibin subunit (alpha, beta(A), and beta(B)) mRNA and inhibin B levels and activation of activin receptors (ACVRs) and Smad signaling pathway in hGL cells obtained from women undergoing in vitro fertilization and cultured with and without activin A treatment after GDF9-targeting small interfering RNA transfection. GDF9, inhibin subunits, ACVR2B/1B and Smad2/3/4/7 mRNA and/or protein levels, Smad phosphorylation, and inhibin B were assessed with RT-PCR, immunoblotting, and ELISA. Data were analyzed by ANOVA followed by Tukey's test. RESULTS: GDF9 was detected as mRNA and protein in hGL cells and protein in follicular fluid from all 11 patients tested. Reduced endogenous GDF9 expression after targeting small interfering RNA transfection was associated with decreased ACVR2B/1B and Smad2/3/4 but increased inhibitory Smad7 mRNA and protein levels and, consequently, reduced activin A-induced beta(B)-subunit mRNA and inhibin B levels. CONCLUSIONS: We report here for the first time autocrine roles for endogenous GDF9 in hGL cells in enhancing activin A-induced beta(B)-subunit mRNA and inhibin B levels via key components of the activin signaling pathway. However, the relative contributions of GDF9 in granulosa cells vs. oocyte as autocrine/paracrine regulators of beta(B)-subunit and inhibin B production in normal and abnormal human ovarian functions remain to be determined.

Growth differentiation factor 9 enhances activin a-induced inhibin B production in human granulosa cells.

Activin A or growth differentiation factor 9 (GDF9) alone can increase beta(B)-mRNA level in human granulosa-lutein cells from women undergoing in vitro fertilization, but their potential interactions and related cell signaling pathways involved are unknown. We therefore compared inhibin subunit and inhibin levels and activation of activin receptors (ACVRs) and Smad signaling pathway in these human granulosa-lutein cells with and without GDF9 and/or activin A treatment. Inhibin subunit (alpha, beta(A), beta(B)), ACVR, and Smad2/3/4/7 mRNA levels, inhibin A and B production, and Smad phosphorylation were assessed by real-time RT-PCR, ELISA, and immunoblotting, respectively. Data were analyzed by ANOVA followed by Tukey's test. Activin A (1-50 ng/ml) or GDF9 (1-200 ng/ml) alone had only little stimulatory effects on alpha- and beta(A)-mRNA levels. In contrast, GDF9 could stimulate beta(B)-subunit levels but to a lesser degree than the dose- and time-dependent effects of activin A. Compared with untreated cells, GDF9 pretreatment for 24 h significantly enhanced activin A-induced beta(B)-mRNA levels, inhibin B secretion, and Smad2/3 phosphorylation (effects attenuated by bone morphogenetic protein receptor 2 extracellular domain, a GDF9 antagonist); and induced ACVR2B/1B and Smad2/3 but reduced Smad7 (an inhibitory Smad) mRNA levels. We report here for the first time that GDF9 enhances cell response to activin A by modulating key components of the activin signaling pathway in regulating inhibin subunits and hence inhibin B production in human granulosa-lutein cells.