Stable over-expression of PPARβ/δ and PPARγ to examine receptor signaling in human HaCaT keratinocytes

Abstract

Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) function and receptor cross-talk with other nuclear receptors, including PPARγ and retinoic acid receptors (RARs), was examined using stable human HaCaT keratinocyte cell lines over-expressing PPARβ/δ or PPARγ. Enhanced ligand-induced expression of two known PPAR target genes, adipocyte differentiation-related protein (ADRP) and angiopoietin-like protein 4 (ANGPTL4), was found in HaCaT keratinocytes over-expressing PPARβ/δ or PPARγ. Over-expression of PPARβ/δ did not modulate the effect of a PPARγ agonist on up-regulation of ADRP or ANGPTL4 mRNA in HaCaT keratinocytes. All-trans retinoic acid (atRA) increased expression of a known RAR target gene, yet despite a high ratio of fatty acid binding protein 5 (FABP5) to cellular retinoic acid binding protein II, did not increase expression of ANGPTL4 or 3-phosphoinositide-dependent-protein kinase 1 (PDPK1), even in HaCaT keratinocytes expressing markedly higher levels of PPARβ/δ. While PPARβ/δ-dependent attenuation of staurosporine- or UVB-induced poly (ADP-ribose) polymerase (PARP) cleavage was not observed, PPARβ/δ- and PPARγ-dependent repression of UVB-induced expression and secretion of inflammatory cytokines was found in HaCaT keratinocytes over-expressing PPARβ/δ or PPARγ. These studies suggest that FABP5 does not transport atRA or GW0742 to PPARβ/δ and promote anti-apoptotic activity by increasing expression of PDPK1, or that PPARβ/δ interferes with PPARγ transcriptional activity. However, these studies demonstrate that stable over-expression of PPARβ/δ or PPARγ significantly increases the efficacy of ligand activation and represses UVB-induced expression of tumor necrosis factor α (TNFα), interleukin 6 (IL6), or IL8 in HaCaT keratinocytes, thereby establishing an excellent model to study the functional role of these receptors in human keratinocytes.

High expression of Fatty Acid‐Binding Protein 5 promotes cell growth and metastatic potential of colorectal cancer cells

“…4A,B). This result is consistent with recent studies showing no changes in the expression of PDPK1 and phosphorylation of AKT in response to GW0742 in human HaCaT keratinocytes and CRC cells 38, 39, 40. In addition, the levels of phosphorylated AKT were unchanged by knockdown of FABP5 (Fig.…”

The ubiquitin ligase MuRF1 regulates PPARα activity in the heart by enhancing nuclear export via monoubiquitination

“…Given the complexity of the PPARα complex and its multiple binding partners (RXR and other nuclear receptors), co-repressors, and co-stimulators, it is not surprising to see opposite activities invoked. For example, co-repression has been described when agonist-bound PPARs attract repressors and when PPAR homodimerization occurs (Fan et al, 2011; Flores et al, 2011; Borland et al, 2011). This sort of feedback mechanism common in physiology has only begun to be touched upon in PPAR biology and rarely in vivo.…”

Peroxisome Proliferator-Activated Receptor β/δ Cross Talks with E2F and Attenuates Mitosis in HRAS-Expressing Cells

“…Cell lysates and supernatants used for Western blot analyses and immunoprecipitations were prepared as previously described (6,18). An NE-PER nuclear protein extraction kit (Thermo Scientific, Rockford, IL) was used to isolate nuclear and cytosol protein.…”

“…Flow cytometry analysis. Cells were stained with bromodeoxyuridine (BrdU) and/or propidium iodide (PI) and analyzed for cell cycle progression as previously described (6,24). The percentage of cells at each phase of the cell cycle Ϯ standard deviation (SD) was determined with FCS Express software.…”