RESUMEN

Aging progresses through the interaction of metabolic processes, including changes in the immune and endocrine systems. Glucocorticoids (GCs), which are regulated by the hypothalamic-pituitary-adrenal (HPA) axis, play an important role in regulating metabolism and immune responses. However, the age-related changes in the secretion mechanisms of GCs remain elusive. Here, we found that corticosterone (CORT) secretion follows a circadian rhythm in young mice, whereas it oversecreted throughout the day in aged mice >18 months old, resulting in the disappearance of diurnal variation. Furthermore, senescent cells progressively accumulated in the zF of the adrenal gland as mice aged beyond 18 months. This accumulation was accompanied by an increase in the number of Ad4BP/SF1 (SF1), a key transcription factor, strongly expressing cells (SF1-high positive: HP). Removal of senescent cells with senolytics, dasatinib, and quercetin resulted in the reduction of the number of SF1-HP cells and recovery of CORT diurnal oscillation in 24-month-old mice. Similarly, administration of a neutralizing antibody against IL1ß, which was found to be strongly expressed in the adrenocortical cells of the zF, resulted in a marked decrease in SF1-HP cells and restoration of the CORT circadian rhythm. Our findings suggest that the disappearance of CORT diurnal oscillation is a characteristic of aging individuals and is caused by the secretion of IL1ß, one of the SASPs, from senescent cells that accumulate in the zF of the adrenal cortex. These findings provide a novel insight into aging. Age-related hypersecretory GCs could be a potential therapeutic target for aging-related diseases.

RESUMEN

As a decoy receptor, soluble ST2 (sST2) interferes with the function of the inflammatory cytokine interleukin (IL)-33. Decreased sST2 expression in colorectal cancer (CRC) cells promotes tumor growth via IL-33-mediated bioprocesses in the tumor microenvironment. In this study, we discovered that hypoxia reduced sST2 expression in CRC cells and explored the associated molecular mechanisms, including the expression of key regulators of ST2 gene transcription in hypoxic CRC cells. In addition, the effect of the recovery of sST2 expression in hypoxic tumor regions on malignant progression was investigated using mouse CRC cells engineered to express sST2 in response to hypoxia. Our results indicated that hypoxia-dependent increases in nuclear IL-33 interfered with the transactivation activity of GATA3 for ST2 gene transcription. Most importantly, hypoxia-responsive sST2 restoration in hypoxic tumor regions corrected the inflammatory microenvironment and suppressed tumor growth and lung metastasis. These results indicate that strategies targeting sST2 in hypoxic tumor regions could be effective for treating malignant CRC.

Asunto(s)

Neoplasias Colorrectales , Interleucina-33 , Animales , Ratones , Interleucina-33/metabolismo , Regulación hacia Abajo , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Núcleo Celular/metabolismo , Neoplasias Colorrectales/genética , Microambiente Tumoral , Factor de Transcripción GATA3/metabolismo

RESUMEN

Long noncoding RNAs (lncRNAs) are important tissue-specific regulators of gene expression, and their dysregulation can induce aberrant gene expression leading to various pathological conditions, including cancer. Although many lncRNAs have been discovered by computational analysis, most of these are as yet unannotated. Herein, we describe the nature and function of a novel lncRNA detected downstream of the human parathyroid hormone (PTH) gene in both extremely rare ectopic PTH-producing retroperitoneal malignant fibrous histiocytoma and parathyroid tumors with PTH overproduction. This novel lncRNA, which we have named "PTH-AS," has never been registered in a public database, and here, we investigated for the first time its exact locus, length, transcription direction, polyadenylation, and nuclear localization. Microarray and Gene Ontology analyses demonstrated that forced expression of PTH-AS in PTH-nonexpressing human breast cancer T47D cells did not induce the ectopic expression of the nearby PTH gene but did significantly upregulate Janus kinase-signal transducer and activator of transcription pathway-related genes such as cancer-promoting interferon-related DNA damage resistance signature (IRDS) genes. Importantly, we show that PTH-AS expression not only enhanced T47D cell invasion and resistance to the DNA-damaging drug doxorubicin but also promoted lung metastasis rather than tumor growth in a mouse xenograft model. In addition, PTH-AS-expressing T47D tumors showed increased macrophage infiltration that promoted angiogenesis, similar to IRDS-associated cancer characteristics. Although the detailed molecular mechanism remains imperfectly understood, we conclude that PTH-AS may contribute to tumor development, possibly through IRDS gene upregulation.

Asunto(s)

Neoplasias de la Mama , ARN Largo no Codificante , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Daño del ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Interferones/metabolismo , Ratones , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo

RESUMEN

Recently, it was reported that 25(OH)D3 (25D3) has physiological bioactivity in certain tissues derived from Cyp27b1 knockout mice. To investigate the function of 25D3 in the kidney as an informational crossroad of various calciotropic substances, we employed the CRISPR-Cas9 system to knock out Cyp27b1 in the mouse renal distal tubular mDCT cell line. Unlike the previously reported mice in which Cyp27b1 was targeted systemically, Cyp27b1 knockout mDCT cells did not produce any measurable 1α,25(OH)2D3 (1,25D3) after 25D3 administration. As was seen with treatment of Cyp27b1 knockout mDCT cells with ≥10-8 M of 1,25D3, the administration of 10-7 M of 25D3 translocated the vitamin D3 receptor (VDR) into the nucleus and promoted the expression of the representative 1,25D3-responsive gene Cyp24a1. The exhaustive target gene profiles of 25D3 were similar to those of 1,25D3. Subsequently, we confirmed that 25D3 induced the expression of the calcium reabsorption-related gene calbindin-D9K, in a way similar to 1,25D3. We also found that 1,25D3 and 25D3 induced the expression of the megalin gene. A chromatin immunoprecipitation assay identified two vitamin D response elements in the upstream region of the megalin gene that seemed to contribute to its expression. Together, we surmise that the ability of 25D3 to stimulate VDR target genes may provide a novel perspective for its role in certain tissues.

Asunto(s)

25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Receptores de Calcitriol/genética , Vitamina D/análogos & derivados , Vitamina D/genética , Animales , Calcio/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Túbulos Renales/efectos de los fármacos , Túbulos Renales/metabolismo , Ratones , Ratones Noqueados , Proteína G de Unión al Calcio S100/genética , Proteína G de Unión al Calcio S100/metabolismo , Vitamina D/metabolismo , Vitamina D/farmacología , Vitamina D3 24-Hidroxilasa/genética

RESUMEN

Glucocorticoid production is regulated by adrenocorticotropic hormone (ACTH) via the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway in the adrenal cortex, but the changes in steroidogenesis associated with aging are unknown. In this study, we show that cell-autonomous steroidogenesis is induced by non-ACTH- mediated genotoxic stress in human adrenocortical H295R cells. Low-dose etoposide (EP) was used to induce DNA damage as a genotoxic stress, leading to cellular senescence. We found that steroidogenesis was promoted in cells stained with γH2AX, a marker of DNA damaged cells. Among stress-associated and p53-inducible genes, the expression of GADD45A and steroidogenesis-related genes was significantly upregulated. Immunofluorescence analysis revealed that GADD45A accumulated in the nuclei. Metabolite assay using cultured media showed that EP-treated cells were induced to produce and secrete considerable amounts of glucocorticoid. Knockdown of GADD45A using small interfering RNA markedly inhibited the EP-induced upregulation of steroidogenesis-related gene expression, and glucocorticoid production. A p38MAPK inhibitor, but not a PKA inhibitor, suppressed EP-stimulated steroidogenesis. These results suggest that DNA damage itself promotes steroidogenesis via one or more unprecedented non-ACTH-mediated pathway. Specifically, GADD45A plays a crucial role in the steroidogenic processes triggered by EP-stimulated genotoxic stress. Our study sheds new light on an alternate mechanism of steroidogenesis in the adrenal cortex.

Asunto(s)

Corteza Suprarrenal/citología , Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Etopósido/farmacología , Proteínas Nucleares/metabolismo , Esteroides/biosíntesis , Células Cultivadas , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

Recently, the antiproliferative action of 1,25(OH)2D3 (1,25D3), an active metabolite of vitamin D3, in the management of prostate cancer has been argued rigorously. In this study, we found that at a physiological concentration, 25(OH)D3 (25D3), the precursor of 1,25D3 and an inactive form of vitamin D because of its much weaker binding activity to the vitamin D receptor (VDR) compared with 1,25D3, had a gene expression profile similar to that of 1,25D3 in prostate cancer LNCaP cells. By immunocytochemistry, western blotting, and CYP27B1 and/or VDR knockdown by small interfering RNAs, we found that 10-7 M 25D3, which is within its uppermost physiological concentration in the bloodstream, induced VDR nuclear import and robustly activated its target genes in the virtual absence of CYP27B1 expression. Comprehensive microarray analyses verified 25D3 bioactivity, and we found that 25D3 target gene profiles largely matched those of 1,25D3, while the presence a small subset of 25D3- or 1,25D3-specific target genes was not excluded. These results indicated that 25D3 shares bioactivity with 1,25D3 without conversion to the latter. Metallothionein 2A was identified as a 1,25D3-specific repressive target gene, which might be a prerequisite for 1,25D3, but not 25D3, to exert its anti-proliferative action in LNCaP cells.

Asunto(s)

Calcifediol/farmacología , Calcitriol/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de la Próstata/genética , Transcriptoma/efectos de los fármacos , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Calcifediol/química , Calcitriol/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Humanos , Hidroxilación , Masculino , Metalotioneína , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Vitaminas/farmacología

RESUMEN

Thus far, only 23 cases of the ectopic production of parathyroid hormone (PTH) have been reported. We have characterized the genome-wide transcription profile of an ectopic PTH-producing tumor originating from a retroperitoneal histiocytoma. We found that the calcium-sensing receptor (CaSR) was barely expressed in the tumor. Lack of CaSR, a crucial braking apparatus in the presence of both intraparathyroid and, probably, serendipitous PTH expression, might contribute strongly to the establishment and maintenance of the ectopic transcriptional activation of the PTH gene in nonparathyroid cells. Along with candidate drivers with a crucial frameshift mutation or copy number variation at specific chromosomal areas obtained from whole exome sequencing, we identified robust tumor-specific cytochrome P450 family 24 subfamily A member 1 (CYP24A1) overproduction, which was not observed in other non-PTH-expressing retroperitoneal histiocytoma and parathyroid adenoma samples. We then found a 2.5-kb noncoding RNA in the PTH 3'-downstream region that was exclusively present in the parathyroid adenoma and our tumor. Such a co-occurrence might act as another driver of ectopic PTH-producing tumorigenesis; both might release the control of PTH gene expression by shutting down the other branches of the safety system (e.g., CaSR and the vitamin D3-vitamin D receptor axis).

RESUMEN

Estrogen receptors (ER) are important transcription factors to relay signals from estrogen and to regulate proliferation of some of breast cancers. The cycling of estrogen-induced DNA binding and ubiquitin-linked proteolysis of ER potentiates ER-mediated transcription. Indeed, several transcriptional coactivators for ER-dependent transcription ubiquitinate ER. Histone acetyltransferase (HAT) Hbo1/KAT7/MYST2, involved in global histone acetylation, DNA replication, transcription, and cellular proliferation, promotes proteasome-dependent degradation of ERα through ubiquitination. However, molecular mechanism for ubiquitination of ERα by Hbo1 is unknown. Here we report the intrinsic ubiquitin E3 ligase activity of Hbo1 toward the ERα. The ligand, estradiol-17ß, inhibited E3 ligase activity of Hbo1 for ERα in vitro, whereas hyperactive ERα mutants from metastatic breast cancers resistant to hormonal therapy, were better substrates for ERα ubiquitination by Hbo1. Hbo1 knock-down caused increase in ERα expression. Hbo1 is another ERα coactivator that ubiquitinates ERα.

Asunto(s)

Receptor alfa de Estrógeno/metabolismo , Histona Acetiltransferasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Animales , Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/genética , Humanos , Mutación , Dominios Proteicos , Especificidad por Sustrato

RESUMEN

The production and secretion of adrenocorticotropin, a proopiomelanocortin (POMC)-derived hormone, by corticotrophs in the anterior pituitary, is regulated by corticotrophin-releasing hormone (CRH) and glucocorticoids. We have previously demonstrated that adrenalectomy induces α-tubulin N-acetyltransferase 1 (ATAT1) expression and α-tubulin acetylation in corticotrophs. However, the regulatory mechanism of ATAT1 expression and the function of acetylated microtubules in corticotrophs are unclear. Here, we analyze the effect of CRH or dexamethasone on Atat1 expression in the mouse corticotroph AtT20 cell line. The expression of Atat1 was increased by CRH and decreased by dexamethasone in AtT20 cells. We examined the effect of Atat1 knockdown on the expression of POMC-associated genes and the dexamethasone-induced nuclear translocation of glucocorticoid receptor (GR) by real-time polymerase chain reaction and Western blot analysis, respectively. Atat1 knockdown resulted in a significant increase in the expression of ACTH-producing genes and decreased the dexamethasone-induced nuclear translocation of GR accompanied with a reduction in α-tubulin acetylation. Atat1 overexpression resulted in a significant increase in α-tubulin acetylation and the dexamethasone-induced nuclear translocation of GR. These results suggest that the acetylated microtubules function as the rail-line for the transportation of GR into the nucleus. We conclude that ATAT1 finely tunes the cellular responses of corticotrophs to hormonal stimulation through an intracellular feedback circuit.

Asunto(s)

Acetiltransferasas/metabolismo , Corticotrofos/fisiología , Hemostasis , Sistema Hipotálamo-Hipofisario/fisiología , Sistema Hipófiso-Suprarrenal/fisiología , Acetilación , Acetiltransferasas/genética , Transporte Activo de Núcleo Celular , Hormona Adrenocorticotrópica/genética , Hormona Adrenocorticotrópica/metabolismo , Animales , Línea Celular , Corticotrofos/citología , Hormona Liberadora de Corticotropina/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ratones , Proteínas de Microtúbulos , Sistema Hipófiso-Suprarrenal/citología , Proopiomelanocortina/genética , Proopiomelanocortina/metabolismo , Receptores de Glucocorticoides/metabolismo , Tubulina (Proteína)/metabolismo

RESUMEN

We previously encountered regulatory processes wherein dihydrotestosterone (DHT) exerted its inhibitory effect on parathyroid hormone-related protein (PTHrP) gene repression through the estrogen receptor (ER)α, but not the androgen receptor (AR), in breast cancer MCF-7 cells. Here, we investigated whether such aberrant ligand-nuclear receptor (NR) interaction is present in prostate cancer LNCaP cells. First, we confirmed that LNCaP cells expressed large amounts of AR at negligible levels of ERα/ß or progesterone receptor. Both suppression of PTHrP and activation of prostate-specific antigen genes were observed after independent administration of 17ß-estradiol (E2), DHT, or R5020. Consistent with the notion that the LNCaP AR lost its ligand specificity due to a mutation (Thr-Ala877), experiments with siRNA targeting the respective NR revealed that the AR monopolized the role of the mediator of shared hormone-dependent regulation, which was invariably associated with nuclear translocation of this mutant AR. Microarray analysis of gene regulation by DHT, E2, or R5020 disclosed that more than half of the genes downstream of the AR (Thr-Ala877) overlapped in the LNCaP cells. Of particular interest, we realized that the AR (wild-type [wt]) and AR (Thr-Ala877) were equally responsible for the E2-AR interactions. Fluorescence microscopy experiments demonstrated that both EGFP-AR (wt) and EGFP-AR (Thr-Ala877) were exclusively localized within the nucleus after E2 or DHT treatment. Furthermore, reporter assays revealed that some other cancer cells exhibited aberrant E2-AR (wt) signaling similar to that in the LNCaP cells. We herein postulate the presence of entangled interactions between wt AR and E2 in certain hormone-sensitive cancer cells.

Asunto(s)

Neoplasias de la Mama/metabolismo , Estradiol/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Línea Celular Tumoral , Dihidrotestosterona/farmacología , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Mutación , Proteína Relacionada con la Hormona Paratiroidea/genética , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Promegestona/farmacología , Receptores Androgénicos/genética

RESUMEN

The ectopic expression of the glucose-dependent insulinotropic polypeptide receptor (GIPR) in the human adrenal gland causes significant hypercortisolemia after ingestion of each meal and leads to Cushing's syndrome, implying that human GIPR activation is capable of robustly activating adrenal glucocorticoid secretion. In this study, we transiently transfected the human GIPR expression vector into cultured human adrenocortical carcinoma cells (H295R) and treated them with GIP to examine the direct link between GIPR activation and steroidogenesis. Using quantitative RT-PCR assay, we examined gene expression of steroidogenic related proteins, and carried out immunofluorescence analysis to prove that forced GIPR overexpression directly promotes production of steroidogenic enzymes CYP17A1 and CYP21A2 at the single cell level. Immunofluorescence showed that the transfection efficiency of the GIPR gene in H295R cells was approximately 5%, and GIP stimulation enhanced CYP21A2 and CYP17A1 expression in GIPR-introduced H295R cells (H295R-GIPR). Interestingly, these steroidogenic enzymes were also expressed in the GIPR (-) cells adjacent to the GIPR (+) cells. The mRNA levels of a cholesterol transport protein required for all steroidogenesis, StAR, and steroidogenic enzymes, HSD3ß2, CYP11A1, CYP21A2, and CYP17A1 increased 1.2-2.1-fold in GIP-stimulated H295R-GIPR cells. These changes were reflected in the culture medium in which 1.5-fold increase in the cortisol concentration was confirmed. Furthermore, the levels of adenocorticotropic hormone (ACTH) receptor and ACTH precursor proopiomelanocortin (POMC) mRNA were upregulated 2- and 1.5-fold, respectively. Immunofluorescence showed that ACTH expression was detected in GIP-stimulated H295R-GIPR cells. An ACTH-receptor antagonist significantly inhibited steroidogenic gene expression and cortisol production. Immunostaining for both CYP17A1 and CYP21A2 was attenuated in cells treated with ACTH receptor antagonists as well as with POMC siRNA. These results demonstrated that GIPR activation promoted production and release of ACTH, and that steroidogenesis is activated by endogenously secreted ACTH following GIP administration, at least in part, in H295R cells.

Asunto(s)

Glándulas Suprarrenales/efectos de los fármacos , Hormona Adrenocorticotrópica/metabolismo , Polipéptido Inhibidor Gástrico/farmacología , Hidrocortisona/metabolismo , Receptores de la Hormona Gastrointestinal/metabolismo , Glándulas Suprarrenales/citología , Glándulas Suprarrenales/metabolismo , Hormona Adrenocorticotrópica/genética , Línea Celular , Colforsina/farmacología , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Humanos , Proopiomelanocortina/antagonistas & inhibidores , Proopiomelanocortina/genética , Proopiomelanocortina/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores de la Hormona Gastrointestinal/genética , Esteroide 17-alfa-Hidroxilasa/genética , Esteroide 17-alfa-Hidroxilasa/metabolismo , Esteroide 21-Hidroxilasa/genética , Esteroide 21-Hidroxilasa/metabolismo , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

Nkx2 homeodomain transcription factors are involved in various developmental processes and cell specification: e.g. in mammals, NKX2-1 is essential for thyroid-specific gene expression and thyroid morphogenesis. Among Nkx2 proteins, information is still very limited for Nkx2-4. In the present study, we have identified three distinct cDNAs encoding Nkx2-4 isoforms (Nkx2-4a, -b, and -c) from the rainbow trout thyroid tissue, and characterized their transcriptional properties. The trout Nkx2-4 proteins were all predicted to conserve three characteristic domains: the tinman-like amino terminal decapeptide, the NK2 homeodomain, and the NK2-specific domain, and also share 75-89% amino acid similarity. It was shown by dual luciferase assay that Nkx2-4a and Nkx2-4b, but not Nkx2-4c, significantly activated transcription from a cotransfected rat thyroglobulin (TG) promoter. An electrophoretic mobility shift assay indicated that all the Nkx2-4 isoforms could bind to the TG promoter, implying that the faint transcriptional activity of Nkx2-4c might result from some critical amino acid substitution(s) outside the homeodomain. RT-PCR analysis revealed similar tissue distribution patterns for Nkx2-4a and Nkx2-4b mRNAs. Both mRNAs were expressed abundantly in the thyroid, and weakly in the testis. On the other hand, Nkx2-4c mRNA was detected in the ovary as well as in the thyroid. The expression sites of Nkx2-4c mRNA were localized, by in situ hybridization histochemistry, to the ovarian granulosa cells and to the thyroid follicular cells. The results suggest that in the rainbow trout, Nkx2-4a and Nkx2-4b might play a major role in TG gene transcription whereas Nkx2-4c might have some functions in the ovary as well as the thyroid.

Asunto(s)

Regulación de la Expresión Génica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/genética , Ensayo de Cambio de Movilidad Electroforética , Hibridación in Situ , Datos de Secuencia Molecular , Oncorhynchus mykiss/metabolismo , Filogenia , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Tiroglobulina/metabolismo , Factor Nuclear Tiroideo 1 , Distribución Tisular , Activación Transcripcional

RESUMEN

We have identified two distinct Pax8 (a and b) mRNAs from the thyroid gland of the rainbow trout (Oncorhynchus mykiss), which seemed to be generated by alternative splicing. Both Pax8a and Pax8b proteins were predicted to possess the paired domain, octapeptide, and partial homeodomain, while Pax8b lacked the carboxy-terminal portion due to an insertion in the coding region of the mRNA. RT-PCR analysis showed each of Pax8a and Pax8b mRNAs to be abundantly expressed in the thyroid and kidney. In situ hybridization histochemistry further detected the expression of Pax8 mRNA in the epithelial cells of the thyroid follicles of the adult trout and in the thyroid primordial cells of the embryo. The functional properties of Pax8a and Pax8b were investigated by dual luciferase assay. The transcriptional regulation by the rat thyroid peroxidase (TPO) promoter was found to be increased by Pax8a, but not by Pax8b. Pax8a further showed synergistic transcriptional activity with rat Nkx2-1 for the human TPO upstream region including the enhancer and promoter. On the other hand, Pax8b decreased the synergistic activity of Pax8a and Nkx2-1. Electrophoretic mobility shift assay additionally indicated that not only Pax8a but also Pax8b can bind to the TPO promoter and enhancer, implying that the inhibitory effect of Pax8b might result from the lack of the functional carboxy-terminal portion. Collectively, the results suggest that for the trout thyroid gland, Pax8a may directly increase TPO gene expression in cooperation with Nkx2-1 while Pax8b may work as a non-activating competitor for the TPO transcription.

Asunto(s)

Empalme Alternativo , Autoantígenos/genética , Proteínas de Peces/metabolismo , Regulación de la Expresión Génica/fisiología , Yoduro Peroxidasa/genética , Proteínas de Unión a Hierro/genética , Oncorhynchus mykiss/genética , Factores de Transcripción Paired Box/genética , Glándula Tiroides/metabolismo , Secuencia de Aminoácidos , Animales , Autoantígenos/metabolismo , Secuencia de Bases , Clonación Molecular , Ensayo de Cambio de Movilidad Electroforética , Proteínas de Peces/genética , Hibridación in Situ , Yoduro Peroxidasa/metabolismo , Proteínas de Unión a Hierro/metabolismo , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oncorhynchus mykiss/crecimiento & desarrollo , Filogenia , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas , ARN Mensajero/genética , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Glándula Tiroides/citología , Factor Nuclear Tiroideo 1 , Distribución Tisular , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional

RESUMEN

The estrogen receptor (ER) is a key molecule for growth of breast cancers. It has been a successful target for treatment of breast cancers. Elucidation of the ER expression mechanism is of importance for designing therapeutics for ER-positive breast cancers. However, the detailed mechanism of ER stability is still unclear. Here, we report that histone acetyltransferase Hbo1 promotes destabilization of estrogen receptor α (ERα) in breast cancers through lysine 48-linked ubiquitination. The acetyltransferase activity of Hbo1 is linked to its activity for ERα ubiquitination. Depletion of Hbo1 and anti-estrogen treatment displayed a potent growth suppression of breast cancer cell line. Hbo1 modulated transcription by ERα. Mutually exclusive expression of Hbo1 and ERα was observed in roughly half of the human breast tumors examined in the present study. Modulation of ER stability by Hbo1 in breast cancers may provide a novel therapeutic possibility.

Asunto(s)

Neoplasias de la Mama/patología , Receptor alfa de Estrógeno/metabolismo , Histona Acetiltransferasas/metabolismo , Ubiquitinación , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular , Antagonistas de Estrógenos/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica , Histona Acetiltransferasas/genética , Humanos , Células MCF-7 , Persona de Mediana Edad , Interferencia de ARN , ARN Interferente Pequeño , Tamoxifeno/farmacología , Transcripción Genética

RESUMEN

We cloned the LIM-homeodomain protein LHX2 as a transcription factor for the porcine follicle-stimulating hormone ß subunit gene (Fshß) by the Yeast One-Hybrid Cloning System using the upstream region of -852/-746 bases (b) from the transcription start site, called Fd2, as a bait sequence. The reporter assay in LßT2 and CHO cells revealed the presence of an LHX2-responsive region other than Fd2. A potential LHX2 binding sequence was confirmed as AATTAAT containing a consensus homeodomain binding core sequence AATT by Systematic Evolution of Ligands by Exponential Enrichment analysis. DNase I footprinting demonstrated three AATTAAT sequences located at regions -835/-829, -818/-812 and -806/-800 b in the Fd2 region and 12 binding sites in the distal and proximal regions mostly containing an AATT-core sequence. RT-PCR analysis of Lhx2 expression during porcine fetal and postnatal pituitary development showed a gradual increase from fetal day (f) 40 to postnatal day (p) 8 followed by a slight decrease to p230, suggesting that LHX2 may play its role largely in the late fetal and postnatal periods. The analyses of Lhx2 expression in pituitary tumor-derived cell lines showed their expressions in cell lines including αT31, LßT2 and others. Since LHX2 was previously identified as a transcription factor for Cga and the in vitro experiments in the present study suggested that LHX2 regulated the expression of Fshß, it is possible that LHX2 controls the synthesis of FSH at the transcription level.

Asunto(s)

Hormona Folículo Estimulante de Subunidad beta/genética , Regulación de la Expresión Génica , Proteínas con Homeodominio LIM/metabolismo , Porcinos/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Células CHO , Línea Celular Tumoral , Clonación Molecular , Cricetinae , Hormona Folículo Estimulante de Subunidad beta/biosíntesis , Proteínas con Homeodominio LIM/genética , Datos de Secuencia Molecular , Hipófisis/crecimiento & desarrollo , Hipófisis/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Transcripción Genética

RESUMEN

The development and differentiation of the pituitary gland progress through spatial and temporal expressions of many transcription factors. Transcription factor HESX1, which begins to be expressed in the Rathke's pouch at the early stage of pituitary development, acts as a transcription repressor. Another transcription factor, PROP1, which is a pituitary-specific factor and important for the determination of the differentiation of pituitary hormone-producing cells, appears later than HESX1 and is assumed to block the action of HESX1. Both factors are members of the homeodomain family, and the amino acid residue at the 50th position of the homeodomain is glutamine (Gln-50). We recently observed that both factors share the same target sequence through different binding profiles. Hence, using random oligonucleotides and an electrophoretic mobility-shift assay, we have examined the DNA-binding preference of HESX1 by a determination of its binding sequence. HESX1 binds as a monomer to a TAATT motif but not to a TAAT motif. In the presence of PROP1, HESX1 develops to bind to an inverted TAAT motif by forming a heterodimer. Thus, the formation of a heterodimer between HESX1 and PROP1 provides a condition in which, in the early pituitary primordium, HESX1 alters its repressive role to an active one by forming a heterodimer with newly appearing PROP1 so that PROP1 finally replaces HESX1 to advance to the middle stage of pituitary development.

Asunto(s)

Secuencias de Aminoácidos , Proteínas de Homeodominio , Hipófisis , Estructura Cuaternaria de Proteína , Animales , Secuencia de Bases , Sitios de Unión , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Hipófisis/anatomía & histología , Hipófisis/crecimiento & desarrollo , Hipófisis/metabolismo , Unión Proteica , Multimerización de Proteína , Proteínas Represoras , Porcinos , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Mutations in the Prop1 gene are responsible for murine Ames dwarfism and human combined pituitary hormone deficiency with hypogonadism. Recently, we reported that PROP1 is a possible transcription factor for gonadotropin subunit genes through plural cis-acting sites composed of AT-rich sequences containing a TAAT motif which differs from its consensus binding sequence known as PRDQ9 (TAATTGAATTA). This study aimed to verify the binding specificity and sequence of PROP1 by applying the method of SELEX (Systematic Evolution of Ligands by EXponential enrichment), EMSA (electrophoretic mobility shift assay) and transient transfection assay. SELEX, after 5, 7 and 9 generations of selection using a random sequence library, showed that nucleotides containing one or two TAAT motifs were accumulated and accounted for 98.5% at the 9th generation. Aligned sequences and EMSA demonstrated that PROP1 binds preferentially to 11 nucleotides composed of an inverted TAAT motif separated by 3 nucleotides with variation in the half site of palindromic TAAT motifs and with preferential requirement of T at the nucleotide number 5 immediately 3' to a TAAT motif. Transient transfection assay demonstrated first that dimeric binding of PROP1 to an inverted TAAT motif and its cognates resulted in transcriptional activation, whereas monomeric binding of PROP1 to a single TAAT motif and an inverted ATTA motif did not mediate activation. Thus, this study demonstrated that dimeric binding of PROP1 is able to recognize diverse palindromic TAAT sequences separated by 3 nucleotides and to exhibit its transcriptional activity.

Asunto(s)

Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Secuencias Invertidas Repetidas/genética , Multimerización de Proteína , Activación Transcripcional/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Secuencia de Consenso , Ensayo de Cambio de Movilidad Electroforética , Proteínas de Homeodominio/química , Datos de Secuencia Molecular , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos/genética , Alineación de Secuencia , Sus scrofa

RESUMEN

This study aimed to identify protein(s) that bind(s) to the highly AT-rich sequence of porcine Fshb promoter region -852/-746 (named Fd2) by the Yeast One-Hybrid Cloning System and finally a paired related homeodomain transcription factor, Prx2, known as a key factor for skeletogenesis was cloned. RT-PCR analysis of fetal and postnatal porcine pituitaries demonstrated that Prx2 starts to be expressed at around fetal days 40-50 just before the beginning of Lhb-expression and that the level of Prx2 increases after birth. Immunohistochemical analysis of the prepubertal porcine pituitary revealed that some Prx2-positive cells overlap some Lh beta-positive cells. Transient transfection assay using non-pituitary CHO cells and pituitary tumor-derived LbetaT2 cells revealed that Prx2 plays a cell-type dependent role in modulation of the Fshb promoter, showing stimulation in CHO cells and repression in LbetaT2 cells via the regions of Fd2 and -596/-239. The binding ability of Prx2 to the regions of Fd2 and -596/-239 was confirmed by electrophoretic mobility shift assay. DNase I footprinting revealed that broad regions of Fd2 were bound by Prx2 and that -596/-239 contained seven Prx2-binding sites. The SELEX method using a random N15-mer oligonucleotide pool demonstrated that Prx2 monomer binds to a TAATT motif, which is present in Fd2 and -596/-239. However, the binding of Prx2 to TAATT with a single molecule and its inverted repeat with two molecules could not induce transcriptional activation, indicating that the Prx2-dependent transcriptional modulation demonstrated in cultured cells is not introduced by Prx2 alone. Thus, this study demonstrated for the first time that Prx2 is expressed in the pituitary gland and at least in a part of gonadotropes in which Prx2 may play a role in repression of the Fshb gene.

Asunto(s)

Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hipófisis/embriología , Hipófisis/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Clonación Molecular , Cricetinae , Cricetulus , Ensayo de Cambio de Movilidad Electroforética , Femenino , Hormona Folículo Estimulante/genética , Hormona Folículo Estimulante/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Embarazo , Regiones Promotoras Genéticas/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Transfección , Técnicas del Sistema de Dos Híbridos

RESUMEN

The aim of this study was to characterize the promoter activity of the porcine pituitary glycoprotein hormone common alpha gene (Cga) promoter (-1059/+12) and the role of LIM homeodomain transcription factor Lhx3. A transfection assay using Chinese hamster ovary (CHO) cells showed that the -1059/-101 region of the Cga promoter definitely responds to Lhx3 and that the -1059/-240 region exhibits a high basal transcriptional level in a pituitary-derived cell line, LbetaT2. A DNA binding and DNase I footprinting assay demonstrated that Lhx3 has seven binding sites in the -1059/+12 region of Cga, including a pituitary glycoprotein hormone basal element (PGBE) known as a LIM homeodomain factor-binding site. A transfection assay of the sequence of Lhx3-binding sites fused with minimal promoter vector confirmed their Lhx3-dependent stimulations in LbetaT2 cells. RT-PCR analysis of porcine pituitary ontogeny demonstrated that porcine Lhx3 showed striking changes of expression in both sexes during the fetal period but a stable high level of expression after birth. Thus, the porcine Cga promoter is regulated by Lhx3 through seven sites in the distal and proximal regions.

Asunto(s)

Regulación de la Expresión Génica , Hormonas Glicoproteicas de Subunidad alfa/biosíntesis , Hormonas Glicoproteicas de Subunidad alfa/genética , Proteínas de Homeodominio/biosíntesis , Animales , Sitios de Unión , Células CHO , Línea Celular , Línea Celular Tumoral , Cricetinae , Cricetulus , ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Proteínas con Homeodominio LIM , Ratones , Estructura Terciaria de Proteína , Porcinos , Factores de Transcripción

RESUMEN

Prophet of PIT1 (PROP1) is a pituitary-specific factor and responsive gene for the combined pituitary hormone deficiency in Ames dwarf mice and human patients. Our immunohistochemical studies demonstrated that PROP1 is consistently expressed in SOX2-expressing stem/progenitor cells in the rat pituitary from embryonic (E) to postnatal periods. At E13.5, all the cells in Rathke's pouch, the primordium of the pituitary, express PROP1. Afterward, PROP1-positive cells localize along the marginal cell layer, a putative stem cell niche in the pituitary, and stratify in the parenchyma of the anterior pituitary. In the embryonic period, PROP1 coexists transiently with PIT1, which is the anterior pituitary-specific factor and is a target of PROP1, but not any hormones. Thus, the present results imply a regulatory role of PROP1 not only in pituitary organogenesis but also in conversion of PIT1-lineage cells.

Asunto(s)

Proteínas de Homeodominio/metabolismo , Organogénesis , Hipófisis/embriología , Factores de Transcripción SOXB1/metabolismo , Nicho de Células Madre/embriología , Factor de Transcripción Pit-1/metabolismo , Animales , Linaje de la Célula , Humanos , Ratones , Hipófisis/citología , Hipófisis/metabolismo , Ratas , Nicho de Células Madre/metabolismo