No abstract

Tributyltin (TBT) is found in human blood and other tissues and thus is of considerable concern as to its effects on human health. Previous studies have demonstrated that TBT has detrimental effects on immune function. Recently, we found that exposures to TBT caused increased secretion of two important proinflammatory cytokines, tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ). Elevation of either of these cytokines has the potential to cause chronic inflammation, which is an important factor in a number of diseases including cancer. The current study examined the mechanism of TBT-induced elevations of TNFα and IFNγ secretion and found that the p38 mitogen-activated protein kinase pathway was essential to the ability of TBT to stimulate secretion. Additionally, this study demonstrated that increased secretion of these cytokines was due to TBT-induced increases in their overall synthesis, rather than simply being due to an increase in the release of already formed proteins. The TBT-induced increases in synthesis were evident within 6 hours of exposure. The p38 mitogen-activated protein kinase pathway is also necessary for the TBT-induced increases in both TNFα and IFNγ synthesis. The role of increased transcription of TNFα and IFNγ mRNA in response to TBT exposures as a possible explanation for the increased synthesis of these cytokines was also examined. It was found that increased mRNA levels did not appear to explain fully the increases in either TNFα or IFNγ synthesis. Thus, TBT is able to increase secretion of two important proinflammatory cytokines by increasing their synthesis.

Collagen, the most abundant protein in humans, is the primary component of the extracellular matrix. Collagen is also a critical reservoir of proline that, along with hydroxyproline, comprise one‐fourth of the amino acid composition of collagen. During collagen turnover, sequential action of matrix metalloproteinases and peptidases convert collagen into dipeptides. In the subsequent final rate‐limiting step, prolidase (PEPD) hydrolyzes dipeptides containing C‐terminal proline or hydroxyproline into the constituent amino acids, which are then recycled towards collagen synthesis. Due to its critical role in collagen turnover and protein metabolism, prolidase is a key player in several physiological and pathological processes, including wound healing, cell proliferation, inflammation, and carcinogenesis. Consequently, alterations in prolidase expression can profoundly affect normal physiological processes and cause pathological changes. The goal of this study is to narrow down such knowledge gaps, especially, in our understanding of the regulation of PEPD transcription. First, towards determining a functionally active promoter region of PEPD (PEPDp), we cloned a 1587 base pair (bp) DNA region—spanning 1537 bp upstream, and 50 bp downstream, of the transcription start site (TSS)—of PEPD gene into a firefly luciferase reporter plasmid. We confirmed that the PEPDp is functionally active by demonstrating its ability to robustly drive the luciferase expression in the HEK293T cell line. Second, we sought to determine the minimal region of PEPDp that is sufficient and necessary to drive gene expression. We subcloned two progressively shorter deletion fragments of PEPDp—537 bp and 387 bp long into the firefly luciferase reporter system and then compared their promoter activity with that of the full‐length PEPDp. Our findings thus indicate that the lack of any cancellation in the promoter activity of the PEPDp deletion fragments points out that the functionally active region of PEPDp spans ~350 bp upstream and 50 bp downstream of the transcriptional start site (TSS), thereby advancing our knowledge of the molecular mechanisms regulating prolidase expression. Support or Funding Information This work is partly supported by National Institutes of Health (NIH) Grants DA037779 and MD007586 (to Jui Pandhare) and DA024558, DA30896, DA033892, DA021471, AI22960 and MD007586 (to Chandravanu Dash). Ireti Eni‐aganga is supported by RISE Grant. The work is also supported by the RCMI Grant G12MD007586, the Vanderbilt CTSA Grant UL1RR024975, the Meharry Translational Research Center (MeTRC) CTSA grant (U54 RR026140 from NCRR/NIH, the U54 Grant MD007593 from NIMHD/NIH, and the Tennessee Center for AIDS Research (P30 AI110527).