Jaideep Chaudhary

The Helix-Loop-Helix Inhibitor of Differentiation (ID) Proteins Induce Post-Mitotic Terminally Differentiated Sertoli Cells to Re-Enter the Cell Cycle and Proliferate

Abstract Prior to puberty the Sertoli cells undergo active cell proliferation, and at the onset of puberty they become a terminally differentiated postmitotic cell population that support spermatogenesis. The molecular mechanisms involved in the postmitotic block of pubertal and adult Sertoli cells are unknown. The four known helix-loop-helix ID proteins (i.e., Id1, Id2, Id3, and Id4) are considered dominant negative regulators of cellular differentiation pathways and act as positive regulators of cellular proliferation. ID proteins are expressed at low levels by postpubertal Sertoli cells and are transiently induced by serum. The hypothesis tested was that ID proteins can induce a terminally differentiated postmitotic Sertoli cell to reenter the cell cycle if they are constitutively expressed. To test this hypothesis, ID1 and ID2 were stably integrated and individually overexpressed in postmitotic rat Sertoli cells. Overexpression of ID1 or ID2 allowed postmitotic Sertoli cells to reenter the cell cycle and undergo mitosis. The cells continued to proliferate even after 300 cell doublings. The functional markers of Sertoli cell differentiation such as transferrin, inhibin alpha, Sert1, and androgen binding protein (ABP) continued to be expressed by the proliferating Sertoli cells, but at lower levels. FSH receptor expression was lost in the proliferating Sertoli cell-Id lines. Some Sertoli cell genes, such as cyclic protein 2 (cathepsin L) and Sry-related HMG box protein-11 (Sox11) increase in expression. At no stage of proliferation did the cells exhibit senescence. The expression profile as determined with a microarray protocol of the Sertoli cell-Id lines suggested an overall increase in cell cycle genes and a decrease in growth inhibitory genes. These results demonstrate that overexpression of ID1 and ID2 genes in a postmitotic, terminally differentiated cell type have the capacity to induce reentry into the cell cycle. The observations are discussed in regards to potential future applications in model systems of terminally differentiated cell types such as neurons or myocytes.

Suppression of c-Fos gene transcription with malignant transformation of human bronchial epithelial cells.

The Activator Protein-1 (AP-1) complex is a dimeric transcription factor composed of fos and jun proteins that regulates cellular growth and differentiation. We previously demonstrated a reduction in basal AP-1 transcriptional activity associated with the malignant transformation of human bronchial epithelial (HBE) cells that was, in part, a consequence of decreased c-fos expression. In this study, we investigated the mechanisms underlying the reduction in c-fos expression associated with the malignant transformation of HBE cells. c-Fos gene transcription was lower in tumorigenic HBE cells than in normal HBE cells, and the reduction in transcription involved c-fos gene promoter elements from −327 to +40. DNaseI footprinting and band shift analyses of motifs within this c-fos promoter region, including a cyclic AMP response element (CRE), serum response element (SRE), sis-inducible element (SIE), and a YY1 site, revealed that binding to these motifs was greater in tumorigenic HBE cells than in normal HBE cells. Site-directed mutagenesis of the CRE partially relieved the repression of c-fos promoter activity in tumorigenic HBE cells. Further, the activity of the Jun N-terminal Kinase (JNK)-dependent pathway, which was a positive regulator of the c-fos promoter, was greater in normal HBE cells than in tumorigenic HBE cells. These findings demonstrate a transcriptionally-mediated suppression of c-fos gene expression associated with the malignant transformation of HBE cells. The decreased activity of the c-fos promoter in tumorigenic 1170I cells appeared to involve suppression through a CRE site and reduced activation by JNK-dependent pathways.

Role of the Transcriptional Coactivator CBP/p300 in Linking Basic Helix-Loop-Helix and CREB Responses for Follicle-Stimulating Hormone-Mediated Activation of the Transferrin Promoter in Sertoli Cells

Abstract Sertoli cells are the epithelial cells responsible for the onset of pubertal development and the maintenance of spermatogenesis in the adult. Transferrin is one of the major secretory products expressed by differentiated Sertoli cells. Investigation of the transcriptional control of transferrin gene expression provides insight regarding the regulation of Sertoli cell differentiation. The optimal activation of the mouse transferrin promoter (mTf) by FSH requires the synergistic actions of the cAMP response element-binding protein (CREB) binding to the cAMP response element-like proximal region II (PRII) and the basic helix-loop-helix (bHLH) binding to the E-box. Proximal region II alone is sufficient for cAMP-mediated activation. The proximity of the PRII and E-box (220 base pairs apart) suggests the possibility of interaction between CREB and bHLH proteins. Such an interaction can be mediated by transcriptional integrators such as CREB-binding protein (CBP) and/or p300 and may stabilize the binding of trans-acting factors to their respective cis-elements. Such an interaction may also provide a mechanism for cell-specific promoter activation. The hypothesis tested in this study was that CBP/p300 is required for the synergistic activation of the transferrin promoter involving PRII and E-box through the formation of a ternary complex. In the Sertoli cells, both CBP and p300 proteins are expressed. The effect of CBP/p300 on transferrin promoter activation and, hence, Sertoli cell function was studied by using antisense oligonucleotides (AS-oligo). In the presence of CBP/p300 AS-oligo, activity of the FSH-induced mTf-chloramphenicol acetyl transferase (CAT) was significantly lower as compared to the respective controls. Interestingly, AS-oligo had no effect on cAMP-induced activation of the transferrin promoter reporter construct (mTf-CAT). Mutations in the E-box (EB*) significantly reduced the FSH response. The presence of AS-oligo had no further effect on the FSH-mediated activation of the EB*-mTf-CAT construct but reduced cAMP-mediated activation. Mutations in the CRE-like PRII (PRII*) also significantly reduced the FSH response. Activation of the PRII*-mTf-CAT in response to cAMP was completely abolished. The presence of AS-oligo had no further effect on the FSH- or cAMP-mediated activation of the PRII*-mTf-CAT construct. In Sertoli cells, CBP/p300 was coimmunoprecipitated with CREB and the bHLH protein E47. These observations suggest that CBP/p300 appears to be involved in regulating FSH-mediated activation of the transferrin promoter by linking bHLH and CREB activities.

The basic helix‐loop‐helix E2A gene product E47, not E12, is present in differentiating Sertoli cells

Sertoli cells are the epithelial cells required to maintain spermatogenesis in the adult testis. The induction of Sertoli cell differentiation in the embryo promotes testicular development and male sex determination. Previous reports suggest that Sertoli cell differentiation is regulated in part by basic helix loop helix transcription factors. This was based on the observation that the promoters of a number of Sertoli cell‐specific genes contain E‐box response elements and over‐expression of Id, a negatively acting bHLH protein, which down regulates Sertoli cell differentiated functions. The present study investigates the ubiquitously expressed bHLH proteins E12 and E47 in Sertoli cells. E12 and E47 are spliced variants of the E2A gene and are implicated in cell‐specific gene expression as part of a dimeric bHLH complex that interacts with E‐box response elements. Both E12 and E47 bHLH proteins are detected in a wide range of cell types. The E12/E47 cDNA product was cloned from Sertoli cells and sequenced. The sequence of the E2A gene product was found to be highly similar to E47, with only two amino acid differences. Characterization of the E2A transcripts suggested that Sertoli cells express E47. Interestingly, using specific polymerase chain reaction (PCR) primers, Sertoli cells were found to only express E47 and not E12. However, E12 was present in the RNA samples obtained from whole testis containing a mixture of cell types. Hormones were found to have no influence on the expression of E47 by Sertoli cells. No other cell system studied to date has been shown to specifically express E47. The results are discussed in relation to the possibility that Sertoli cells may express a more cell‐specific bHLH protein that can preferentially dimerize with E47, and/or that E47 homodimers may be transcriptionally active in Sertoli cells. Mol. Reprod. Dev. 52:1–8, 1999.

Comparative sequence analysis of the mouse and human transferrin promoters: hormonal regulation of the transferrin promoter in Sertoli cells.

Cell‐specific expression of the iron‐binding protein transferrin is in part mediated through the regulation of its promoter. Although all cells require iron from serum transferrin produced by hepatocytes, cells that create a blood barrier such as Sertoli cells in the testis and choroid plexus epithelium in the brain also express the transferrin gene to provide iron to cells sequestered within the serum‐free environment. The current study provides a complete sequence of the 3‐kb mouse transferrin promoter and makes a comparison with the sequence available for the human transferrin promoter. Conserved regulatory elements between these two species are identified and speculated to be potentially important response elements for the regulation of the transferrin gene. The proximal 90 bp of the mouse and human transferrin promoter was found to be 80% homologous. The previously identified protected regions in the proximal human promoter also were conserved in the mouse transferrin promoter. Our sequence analysis data revealed that an E‐box response element is also conserved between mouse and human promoters. Deletion mutants of the mouse transferrin promoter were generated in CAT reporter constructs to study the regulation of the transferrin promoter in Sertoli cells. As in the case of the human transferrin promoter, the mouse 581‐bp proximal transferrin promoter was sufficient to obtain basal expression. A putative cyclic AMP response element (CRE) in the minimal promoter may be needed for follicle‐stimulating hormone (FSH) actions mediated via cyclic AMP. Interestingly, other regulatory agents such as the testicular paracrine factor PModS used elements in the upstream region. A repressor was identified 2.5 kb upstream from the start site of translation. Combined observations suggest for the first time that a minimal promoter is sufficient for basal transcription, but the upstream regions of the promoter are needed for the hormonal regulation of the transferrin gene in Sertoli cells. Conserved response elements between the mouse and human sequences identify potentially important regulatory elements of the promoter and are discussed. Mol. Reprod. Dev. 50:273–283, 1998.

Transcription Factors in Sertoli Cells

This chapter discusses the role of transcription factors in regulating Sertoli cell-specific gene expression. Transcription factors are DNA binding proteins ( trans -acting) that bind to DNA regulatory elements located cis to the target genes. Transcription factors are generally considered final targets of signal-transduction pathways. The level of expression of transcription factors and their activities determines whether their target genes are transcribed and to what extent. These regulators of gene expression in turn are tightly regulated by a multitude of signaling pathways influenced by cell–cell interaction, growth factors, hormones, extracellular matrix, and the stage of development. Alterations in the transcriptional regulation of the Sertoli cell determine changes in cellular differentiation and function. The functional and developmental changes associated with Sertoli cell differentiation and functions are a result of stage-specific activation/repression of specific transcription factors. Some of these transcription factors such as Sry and DAX-1are expressed only during a particular stage of development.