Induction of ubiquitin C (UBC) gene transcription is mediated by HSF1: role of proteotoxic and oxidative stress

Abstract

The polyubiquitin gene ubiquitin C ( UBC ) is considered a stress protective gene and is upregulated under various stressful conditions, which is probably a consequence of an increased demand for ubiquitin in order to remove toxic misfolded proteins. We previously identified heat shock elements ( HSE s) within the UBC promoter, which are responsible for heat shock factor ( HSF )1‐driven induction of the UBC gene and are activated by proteotoxic stress. Here, we determined the molecular players driving the UBC gene transcriptional response to arsenite treatment, mainly addressing the role of the nuclear factor‐erythroid 2‐related factor 2 (Nrf2)‐mediated antioxidant pathway. Exposure of HeLa cells to arsenite caused a time‐dependent increase of UBC mRNA , while cell viability and proteasome activity were not affected. Nuclear accumulation of HSF 1 and Nrf2 transcription factors was detected upon both arsenite and MG 132 treatment, while HSF 2 nuclear levels increased in MG 132‐treated cells. Notably, si RNA ‐mediated knockdown of Nrf2 did not reduce UBC transcription under either basal or stressful conditions, but significantly impaired the constitutive and inducible expression of well‐known antioxidant response element‐dependent genes. A chromatin immunoprecipitation assay consistently failed to detect Nrf2 binding to the UBC promoter sequence. By contrast, depletion of HSF 1, but not HSF 2, significantly compromised stress‐induced UBC expression. Critically, HSF 1‐mediated UBC trans ‐activation upon arsenite exposure relies on transcription factor binding to previously mapped distal HSE s, as demonstrated to occur under proteasome inhibition. These data highlight HSF 1 as the pivotal transcription factor that translates different stress signals into UBC gene transcriptional induction.

The Ubiquitin Gene Expression Pattern and Sensitivity to UBB and UBC Knockdown Differentiate Primary 23132/87 and Metastatic MKN45 Gastric Cancer Cells

“…To shed light on the molecular players supporting Ub gene expression in GC cells, we preliminarily evaluated the levels and the intracellular distribution of HSF1, YY1 and SP1 transcription factors in the MKN45 compared to the 23132/87 cell line. Several pieces of evidence in the literature, including our previous studies, indeed reported the involvement of these transcriptional regulators in the expression of the Ub genes, either in basal or stressful conditions [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ].…”

“…While UBA52 and RPS27A are constitutively expressed and contribute with UBB and UBC to fulfill the ubiquitin demand in basal conditions, only UBB and UBC are induced when cells are exposed to threats such as heat shock, oxidative stress and proteotoxic stress [ 19 , 20 , 21 , 32 ]. To determine total Ub concentration, whole cell extracts from both cell lines were treated with the Usp2 deubiquitinating enzyme to convert all conjugated Ub species into monomeric Ub [ 33 ].…”

“…However, TF knockdown did not affect Ub gene transcription, as detected by RTqPCR of cDNA samples derived from silenced GC cells compared to si GFP transfected cells ( Figure 3 I,J). Regarding HSF1, its activity is mainly induced in response to different stressful conditions such as heat shock, oxidative stress or proteotoxic stress [ 19 , 21 ]: this may account for the lack of effect on UBC and UBB gene transcription in HSF1 silenced gastric cancer cells. Instead, the output of YY1 transcription factor silencing was further investigated by RTqPCR analysis of three known direct targets of YY1: Bcl-2-like protein 4 ( BAX ) and Avian myelocitomatosis virus oncogene cellular homolog ( c-MYC ), which are positively regulated by YY1 [ 37 ], and the survivin coding gene, which is repressed by YY1 [ 38 ].…”

“…A role for Specificity Protein 1 (SP1) in the transcriptional regulation of UBC has also been reported [ 14 , 17 , 18 ]. By contrast, Heat Shock Factor 1 (HSF1) is the main transcription factor involved in the upregulation of UBC gene expression under several stress conditions [ 16 , 19 , 20 , 21 ]. In addition, several reports have demonstrated the pro-survival and pro-carcinogenetic role of YY1 [ 22 ], SP1 [ 23 ] and HSF1 [ 24 ] in gastric cancer (GC) development.…”

Imidazo[1,2-b]pyrazole-7-carboxamides Induce Apoptosis in Human Leukemia Cells at Nanomolar Concentrations

“…Early endoplasmic reticulum stress-associated genes, activating transcription factor 3 (ATF3), and DNA damage inducible transcript 3 (GADD153), elevated with chaperons like calreticulin (CALR) and heat shock protein family A (Hsp70) member 1A (HSPA1A) from 12 h of incubation. Other genes associated with differentiation and cell adhesion: Growth differentiation factor 15 (GDF15), galectin-1 (LGALS1), lamin B receptor (LBR) were upregulated at 12 h. Polyubiquitin genes and Bcl family members regulate cell survival and apoptosis [ 28 , 29 ], ubiquitin C has been described as a stress-response gene [ 30 , 31 ] activated at 12 h in MV-4-11 cells. The apoptosis regulator BCL2A1 (Bfl-1/A1) also elevated at 12 and 24 h upon stimulation, which has been described as anti-apoptotic [ 32 ], but also as pro-apoptotic via proteasome mediated turnover or calpain like cleavage [ 33 ].…”