Dietary biotin supplementation increases proliferation pathways in mice testes without affecting serum follicle-stimulating hormone levels and stem cell factor expression.

Supplements containing pharmacological concentrations of biotin are commercially available. The mechanisms by which biotin at pharmacological concentrations exerts its action have been the subject of multiple investigations, particularly for biotin's medicinal potential and wide use for cosmetic purposes. Several studies have reported that biotin supplementation increases cell proliferation; however, the mechanisms involved in this effect have not yet been characterized. In a previous study, we found that a biotin-supplemented diet increased spermatogonia proliferation. The present study was focused on investigating the molecular mechanisms involved in biotin-induced testis cell proliferation. Male BALB/cAnNHsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for eight weeks. Compared with the control group, the biotin-supplemented mice presented augmented protein abundance of the c-kit-receptor and pERK1/2Tyr204 and pAKTSer473, the active forms of ERK/AKT proliferation signaling pathways. No changes were observed in the testis expression of the stem cell factor and in the serum levels of the follicle-stimulating hormone. Analysis of mRNA abundance found an increase in cyclins Ccnd3, Ccne1, Ccna2; Kinases Cdk4, Cdk2; and E2F; and Sp1 & Sp3 transcription factors. Decreased expression of cyclin-dependent kinase inhibitor 1a (p21) was observed but not of Cdkn2a inhibitor (p16). The results of the present study identifies, for the first time, the mechanisms associated with biotin supplementation-induced cell proliferation, which raises concerns about the effects of biotin on male reproductive health because of its capacity to cause hyperplasia, especially because this vitamin is available in large amounts without regulation.

Induction of cell differentiation activates transcription of the Sarco/Endoplasmic Reticulum calcium-ATPase 3 gene (ATP2A3) in gastric and colon cancer cells.

The Sarco/Endoplasmic Reticulum Ca2+ -ATPases (SERCAs), pump Ca2+ into the endoplasmic reticulum lumen modulating cytosolic Ca2+ concentrations to regulate various cellular processes including cell growth. Previous studies have reported a downregulation of SERCA3 protein expression in gastric and colon cancer cell lines and showed that in vitro cell differentiation increases its expression. However, little is known about the transcriptional mechanisms and transcription factors that regulate SERCA3 expression in epithelial cancer cells. In this work, we demonstrate that SERCA3 mRNA is upregulated up to 45-fold in two epithelial cancer cell lines, KATO-III and Caco-2, induced to differentiate with histone deacetylase inhibitors (HDACi) and by cell confluence, respectively. To evaluate the transcriptional elements responding to the differentiation stimuli, we cloned the human ATP2A3 promoter, generated deletion constructs and transfected them into KATO-III cells. Basal and differentiation responsive DNA elements were located by functional analysis within the first -135 bp of the promoter region. Using site-directed mutagenesis and DNA-protein binding assays we found that Sp1, Sp3, and Klf-4 transcription factors bind to ATP2A3 proximal promoter elements and regulate basal gene expression. We showed that these factors participated in the increase of ATP2A3 expression during cancer cell differentiation. This study provides evidence for the first time that Sp1, Sp3, and Klf-4 transcriptionally modulate the expression of SERCA3 during induction of epithelial cancer cell differentiation. © 2016 Wiley Periodicals, Inc.

SMAD3 and SP1/SP3 Transcription Factors Collaborate to Regulate Connective Tissue Growth Factor Gene Expression in Myoblasts in Response to Transforming Growth Factor ß.

Fibrotic disorders are characterized by an increase in extracellular matrix protein expression and deposition, Duchene Muscular Dystrophy being one of them. Among the factors that induce fibrosis are Transforming Growth Factor type ß (TGF-ß) and the matricellular protein Connective Tissue Growth Factor (CTGF/CCN2), the latter being a target of the TGF-ß/SMAD signaling pathway and is the responsible for the profibrotic effects of TGF-ß. Both CTGF and TGF are increased in tissues affected by fibrosis but little is known about the regulation of the expression of CTGF mediated by TGF-ß in muscle cells. By using luciferase reporter assays, site directed mutagenesis and specific inhibitors in C2C12 cells; we described a novel SMAD Binding Element (SBE) located in the 5' UTR region of the CTGF gene important for the TGF-ß-mediated expression of CTGF in myoblasts. In addition, our results suggest that additional transcription factor binding sites (TFBS) present in the 5' UTR of the CTGF gene are important for this expression and that SP1/SP3 factors are involved in TGF-ß-mediated CTGF expression.

Characterization of human collagen XVIII promoter 2: interaction of Sp1, Sp3 and YY1 with the regulatory region and a SNP that increases transcription in hepatocytes.

Different levels of Collagen XVIII expression have been associated with several pathological processes such as cancer, liver fibrosis, diabetic retinopathy and Alzheimer's disease. Understanding the transcriptional regulation of Collagen XVIII might elucidate some pathways related to the progression of these diseases. The promoter 2 of COL18A1 gene is poorly understood and is responsible for the transcription of this gene in several adult tissues such as liver, eyes and brain. This study focused upon characterization of cis-regulatory elements interacting with human COL18A1 promoter 2 and identification of SNPs in this region in different ethnic groups. Our results show that there are five conserved regions (I to V) between human and mouse promoter 2 and that the human COL18A1 core promoter is located between nucleotides -186 and -21. Sp1 and Sp3 bind to conserved regions I and V, while Sp3 and YY1 interact with region II. We have verified that the SNP at position -700 (T>G) is embedded in two common haplotypes, which have different frequencies between European and African descendents. The allele -700G increases transcription and binding for a still unknown transcription factor. SNP -700 affects Sp3 and YY1 interaction with this region, even though it is not part of these transcription factors' predicted binding sites. Therefore, our results show for the first time that Sp3 and YY1 interact with human COL18A1 promoter 2, and that nucleotide -700 is part of a binding motif for a still unknown TF that is involved in the expression of this gene in hepatocytes. In addition, we also confirm the involvement of Sp1 in the regulation of this gene.