Molecular Cloning and Characterization of Porcine Homeodomain Transcription Factor Msx1

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The pituitary is an important endocrine tissue of the vertebrate that produces and secretes many hormones. Accumulating data suggest that several types of cells compose the pituitary, and there is growing interest in elucidating the origin of these cell types and their roles in pituitary organogenesis. Therein, the histogenous cell line is an extremely valuable experimental tool for investigating the function of derived tissue. In this study, we compared gene expression profiles by microarray analysis and real-time PCR for murine pituitary tumor-derived non-hormone-producing cell lines TtT/GF, Tpit/F1 and Tpit/E. Several genes are characteristically expressed in each cell line: Abcg2, Nestin, Prrx1, Prrx2, CD34, Eng, Cspg4 (Ng2), S100β and nNos in TtT/GF; Cxcl12, Raldh1, Msx1 and Twist1 in Tpit/F1; and Cxadr, Sox9, Cdh1, EpCAM and Krt8 in Tpit/E. Ultimately, we came to the following conclusions: TtT/GF cells show the most differentiated state, and may have some properties of the pituitary vascular endothelial cell and/or pericyte. Tpit/F1 cells show the epithelial and mesenchymal phenotypes with stemness still in a transiting state. Tpit/E cells have a phenotype of epithelial cells and are the most immature cells in the progression of differentiation or in the initial endothelial-mesenchymal transition (EMT). Thus, these three cell lines must be useful model cell lines for investigating pituitary stem/progenitor cells as well as organogenesis.

Section: Discussionmentioning

confidence: 99%

Characterization of Murine Pituitary-Derived Cell Lines Tpit/F1, Tpit/E and TtT/GF

Yoshida

Higuchi

Ueharu

et al. 2014

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“…Upstream regions of the rat Cga (NC_005104.4), Lhb (NC_005100.4) and Fshb (NC_005102.4) genes were amplified using specific primer sets. The fragments were ligated into the secreted alkaline phosphatase (SEAP) plasmid vector pSEAP2-Basic (Clontech Laboratories, Palo Alto, CA, USA) as described previously [ 21 , 22 , 23 ]. The resulting reporter vectors contained the following gonadotropin subunit upstream regions: -3793/+37 ( Cga ); -2930/+17 ( Lhb ); and -2824/+28, -2342/+28, -1718/+28, -1499/+28, -1219/+28, -970/+28, -650/+28, -346/+28, -43/+28 ( Fshb ).…”

Section: Methodsmentioning

confidence: 99%

Long-chain unsaturated fatty acids reduce the transcriptional activity of the rat follicle-stimulating hormone β-subunit gene

Moriyama

Yamazaki

Katō

et al. 2016

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Here, we assessed the effects of long-chain fatty acids (LCFAs) and the LCFA receptor agonist GW9508 on the transcription of the gonadotropin subunit genes Cga, Lhb and Fshb because LCFA receptor GPR120 was observed in mouse gonadotropes in our recent study. A transcription assay using LβT2 cells demonstrated that LCFAs, oleic acid, α-linolenic acid, docosahexaenoic acid and palmitate, repressed the expression of Cga, Lhb, and Fshb at concentrations between 50 and 100 µM. On the other hand, treatment with 10 µM unsaturated LCFAs, oleic acid, α-linolenic acid and docosahexaenoic acid, repressed only Fshb expression, while the same dose of a saturated LCFA, palmitate, had no effect on the expression of gonadotropin subunit genes. Furthermore, GW9508 did not affect promoter activity. Next, we examined deletion mutants of the upstream region of Fshb and found that the upstream regulatory region (-2824 to -2343 bp) of Fshb was responsible for the notable repression by 10 µM unsaturated LCFAs. Our results suggest that the upstream region of Fshb is susceptible to unsaturated LCFAs. In addition, unsaturated LCFAs play a role in repressing Fshb expression through the distal -2824 to -2343 bp region, which might be independent of the LCFA receptor GPR120 pathway.

“…The culture and transfection conditions for Chinese Hamster Ovary (CHO) cells (obtained from RIKEN Cell Bank, Ibaraki, Japan) and an immortalized gonadotrope cell line, LβT2 (kindly provided by Dr. PL Mellon), have been described previously [30,31]. (Roche)/well in a 96-well plate.…”

Section: Fshb(-2323/+10) Fshb(-1965/+10) Fshb(-985/+10) Fshb(-596/mentioning

confidence: 99%

Molecular Cloning of Paired Related Homeobox 2 (Prx2) as a Novel Pituitary Transcription Factor

Sugiyama

Ishikawa

Katō

et al. 2009

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Abstract. 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 Lhβ-positive cells. Transient transfection assay using nonpituitary CHO cells and pituitary tumor-derived LβT2 cells revealed that Prx2 plays a cell-type dependent role in modulation of the Fshb promoter, showing stimulation in CHO cells and repression in LβT2 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. Key words: Follicle-stimulating hormone (FSH), Gonadotropin, Pituitary, Prx2, Transcription factor (J. Reprod. Dev. 55: [502][503][504][505][506][507][508][509][510][511] 2009) ollicle-stimulating hormone (FSH), in addition to luteinizing hormone (LH) and thyroid-stimulating hormone (TSH), is a member of the pituitary glycoprotein hormone family consisting of a common α subunit (αGSU) and a noncovalently associated hormone-specific β subunit and plays crucial roles in folliculogenesis in females and spermatogenesis in males. Notably, three gonadotropin subunit genes, Cga, Fshb and Lhb, are expressed in the same pituitary cell, the gonadotrope. Several investigations have been conducted to clarify the specific regulatory mechanism of Cga and two β subunit genes (reviewed in Refs. [1] We have previously observed that plural nuclear proteins specifically bind to the AT-rich sequence of the porcine Fshb promoter region -852/-746 (named Fd2) [10], and have demonstrated that the upstream region -852/+10 containing Fd2 is sufficient for gonadotrope-specific expression [11]. Cloning by the Yeast OneHybrid System using Fd2 as a bait sequence led us to select plural proteins including Prop1 and Lhx2 [12,13]. At the same t...