The hypothalamic hormone gonadotropin-releasing hormone (GnRH) is released in a pulsatile fashion, with its frequency varying throughout the reproductive cycle. Varying pulse frequencies and amplitudes differentially regulate the biosynthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by pituitary gonadotropes. The mechanism by which this occurs remains a major question in reproductive physiology. Previous studies have been limited by lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have overcome this limitation by transfecting the rat pituitary GH3 cell line with rat GnRH receptor (GnRHR) cDNA driven by a heterologous promoter. These cells, when cotransfected with regulatory regions of the common alpha, LH beta, or FSH beta subunit gene fused to a luciferase reporter gene, respond to GnRH with an increase in luciferase activity. Using this model, we demonstrate that different cell surface densities of the GnRHR result in the differential regulation of LH and FSH subunit gene expression by GnRH. This suggests that the differential regulation of gonadotropin subunit gene expression by GnRH observed in vivo in rats may, in turn, be mediated by varying gonadotrope cell surface GnRHR concentrations. This provides a physiologic mechanism by which a single ligand can act through a single receptor to regulate differentially the production of two hormones in the same cell.

Overcoming senescence signals in somatic cells is critical to cellular immortalization and carcinogenesis. High-risk human papillomavirus (HPV) can immortalize epithelial cells in culture through degradation of the retinoblastoma protein by HPV E7 and activation of hTERT transcription, the catalytic subunit of telomerase, by the heterodimer HPV E6/E6-associated protein (E6AP). Recent work in our laboratory identified a novel repressor of hTERT transcription, NFX1-91, which is targeted for ubiquitin-mediated degradation by HPV type 16 (HPV16) E6/E6AP. In contrast, NFX1-123, a splice variant NFX1, increased expression from an hTERT promoter that was activated by HPV16 E6/E6AP. Here, we show that HPV16 E6 bound both NFX1-91 and NFX1-123 through the common central domain of NFX1 in the absence of E6AP. NFX1-123 positively regulated hTERT expression, as its knockdown decreased hTERT mRNA levels and telomerase activity and its overexpression increased telomerase activity. We identified new protein partners of NFX1-123, including several cytoplasmic poly(A) binding proteins (PABPCs) that interacted with NFX1-123 through its N-terminal PAM2 motif, a protein domain characteristic of other PABPC protein partners. Furthermore, NFX1-123 and PABPCs together had a synergistic stimulatory effect on hTERT -regulated reporter assays. The data suggest that NFX1-123 is integral to hTERT regulation in HPV16 E6-expressing epithelial cells and that the interaction between NFX1-123 and PABPCs is critical to hTERT activity.

High-risk human papillomavirus (HPV) E6 protein induces telomerase activity through transcriptional activation of hTERT, the catalytic subunit of telomerase. HPV type 16 (HPV16) E6 interacts with two splice variants of NFX1 to increase hTERT expression. NFX1-91 is a transcriptional repressor of hTERT that is polyubiquitinated and targeted for degradation by HPV16 E6 in concert with E6-associated protein. We previously showed that NFX1-123 augments hTERT expression through binding to cytoplasmic poly(A) binding proteins (PABPCs). In this study, we determined that unlike NFX1-91, NFX1-123 is a cytoplasmic protein that colocalized with PABPCs but does not shuttle with PABPCs between the nucleus and cytoplasm. NFX1-123 requires both its PAM2 motif, with which it binds PABPCs, and its R3H domain, which has putative nucleic acid binding capabilities, to increase hTERT mRNA levels and telomerase activity in keratinocytes expressing HPV16 E6. In keratinocytes expressing HPV16 E6 and overexpressing NFX1-123, there was increased protein expression from in vitro-transcribed RNA fused with the 5′ untranslated region (5′ UTR) of hTERT. This posttranscriptional increase in expression required the PAM2 motif and R3H domain of NFX1-123 as well as the coexpression of HPV16 E6. NFX1-123 bound endogenous hTERT mRNA and increased its stability in HPV16 E6-expressing human foreskin keratinocytes, and NFX1-123 increased the stability of in vitro-transcribed RNA fused with the 5′ UTR of hTERT. Together, these studies describe the first evidence of posttranscriptional regulation of hTERT, through the direct interaction of the cytoplasmic protein NFX1-123 with hTERT mRNA, in HPV16 E6-expressing keratinocytes.

Telomerase extends the repetitive DNA at the ends of linear chromosomes, and it is normally active in stem cells. When expressed in somatic diploid cells, it can lead to cellular immortalization. Human papillomaviruses (HPVs) are associated with and high-risk for cancer activate telomerase through the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT). The expression of hTERT is affected by both high-risk HPVs, E6 and E7. Seminal studies over the last two decades have identified the transcriptional, epigenetic, and post-transcriptional roles high-risk E6 and E7 have in telomerase induction. This review will summarize these findings during infection and highlight the importance of telomerase activation as an oncogenic pathway in HPV-associated cancer development and progression.