title: 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.
authors: Graduate Program in Pharmacology and Toxicology, Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT, 06269-3092, USA.
authors: Vincent P
authors: VP
authors: Ramirez
authors: Michael
authors: M
authors: Stamatis
authors: Anastasia
authors: A
authors: Shmukler
authors: Brian J
authors: BJ
authors: Aneskievich
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D024363:Transcription Initiation Site
chemical_list: D004268:DNA-Binding Proteins; C000619706:HSF1 protein, human; D018840:HSP70 Heat-Shock Proteins; C544598:HSPA6 protein, human; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D014157:Transcription Factors; C070389:HSF2 protein, human