Basal and stress-inducible expression of HSPA6 in human keratinocytes is regulated by negative and positive promoter regions

Abstract

Epidermal keratinocytes serve as the primary barrier between the body and environmental stressors. They are subjected to numerous stress events and are likely to respond with a repertoire of heat shock proteins (HSPs). HSPA6 (HSP70B′) is described in other cell types with characteristically low to undetectable basal expression, but is highly stress induced. Despite this response in other cells, little is known about its control in keratinocytes. We examined endogenous human keratinocyte HSPA6 expression and localized some responsible transcription factor sites in a cloned HSPA6 3 kb promoter. Using promoter 5′ truncations and deletions, negative and positive regulatory regions were found throughout the 3 kb promoter. A region between −346 and −217 bp was found to be crucial to HSPA6 basal expression and stress inducibility. Site-specific mutations and DNA-binding studies show that a previously uncharacterized AP1 site contributes to the basal expression and maximal stress induction of HSPA6. Additionally, a new heat shock element (HSE) within this region was defined. While this element mediates increased transcriptional response in thermally stressed HaCaT keratinocytes, it preferentially binds a stress-inducible factor other than heat shock factor (HSF)1 or HSF2. Intriguingly, this newly characterized HSPA6 HSE competes HSF1 binding a consensus HSE and binds both HSF1 and HSF2 from other epithelial cells. Taken together, our results demonstrate that the HSPA6 promoter contains essential negative and positive promoter regions and newly identified transcription factor targets, which are key to the basal and stress-inducible expression of HSPA6. Furthermore, these results suggest that an HSF-like factor may preferentially bind this newly identified HSPA6 HSE in HaCaT cells.

Cadmium-mediated activation of the HSP90/HSF1 pathway regulated by reactive persulfides/polysulfides

“…This may have been because cadmium strongly induced the HSPA6 gene. We speculate that such a strong induction of the HSPA6 gene is caused by multiple HSEs in the promoter region and other factors such as AP-1 (Ramirez et al, 2015) because cadmium is known to activate AP-1 (Thevenod and Lee, 2013). In our study, HSF1 knockdown suppressed the inducible HSP70 but not its basal expression (Figure 2A), suggesting that HSF1 is essential to the expression of the inducible HSP70.…”

Induction of heat shock proteins in differentiated human neuronal cells following co-application of celastrol and arimoclomol

“…This includes upregulation of HSPA6 (Hsp70B'), a little studied member of the HSPA (Hsp70) family that is present in the human genome, but not in rat and mouse (Chow and Brown 2007;Noonan et al 2007aNoonan et al , b, 2008aChow et al 2010;Ramirez et al 2014). Recently, we have demonstrated a unique feature of HSPA6, namely localization to transcription sites in human neuronal cells during recovery from stress-induced inhibition (Khalouei et al 2014), a feature that is missing in current animal models of neurodegenerative diseases which lack HSPA6.…”

Dynamics of the association of heat shock protein HSPA6 (Hsp70B’) and HSPA1A (Hsp70–1) with stress-sensitive cytoplasmic and nuclear structures in differentiated human neuronal cells

“…In the present study, live imaging and FRAP were utilized to further knowledge of HSPA6 (Hsp70B'), a little studied member of the HSPA (Hsp70) multigene family that is present in the human genome but absent in the genomes of mouse and rat (Chow and Brown 2007;Noonan et al 2007a;Noonan et al 2007b;Noonan et al 2008a;Noonan et al 2008b;Chow et al 2010;Ramirez et al 2015;Deane and Brown 2016). Following thermal stress, changes in the intracellular localization of HSPA6, and the more widely studied HSPA1A (Hsp70-1), were visualized in living differentiated human neuronal SH-SY5Y cells, with FRAP employed to compare the dynamics of the exchange of these two Hsps with stresssensitive cytoplasmic and nuclear structures.…”

“…Heat shock proteins (Hsps) are cellular repair agents that counter the effects of protein misfolding, and their upregulation has been proposed as a potential strategy to counter neurodegenerative disorders (Muchowski and Wacker 2005;Asea and Brown 2008;Pratt et al 2015). The HSPA (Hsp70) family has been widely studied, particularly HSPA1A (Hsp70-1), however HSPA6 (Hsp70B') has received comparatively little attention (Chow and Brown 2007;Noonan et al 2007a;Noonan et al 2007b;Noonan et al 2008a;Noonan et al 2008b;Chow et al 2010;Ramirez et al 2015;Deane and Brown 2016). Interestingly, HSPA6 is present in the human genome and absent in the genomes of mouse and rat.…”