Activation profiles of HSPA5 during the glomerular mesangial cell stress response to chemical injury

Abstract

Environmental injury has been associated with endoplasmic reticulum (ER) stress, a response characterized by activation of the unfolded protein response, proteasomal degradation of proteins, and induction of HSPA5, also known as GRP78 or BiP. Although HSPA5 has been implicated in the stress response to environmental injury in several cell types, its role in the glomerular ER stress response is unknown. In this study, we evaluated HSPA5 activation profiles in rat glomerular mesangial cells (rGMCs) challenged with heavy metals (HgCl2 or Pb2+ acetate) or polycyclic aromatic hydrocarbons (PAHs, ie, benzo(a)pyrene [BaP]). Challenge of rGMCs with 1 or 10 microM HgCl2 or Pb2+ acetate increased HSPA5 mRNA and protein levels. The induction response was sensitive to transcriptional and translational inhibition by actinomycin D (AD) and cyclohexamide, respectively. HSPA5 mRNA was induced by 3 microM BaP in an AD-sensitive manner, but this response was unaffected by the presence of heavy metals. A promoter construct containing sequences that mediate thapsigargin (TH) inducibility of the HSPA5 promoter was refractory to both heavy metals and BaP. The HSPA5 induction response in rGMCs is conserved because it was reproduced with fidelity in immunolocalization experiments of HSPA5 protein in M15 and HEK293 cells in embryonic lines of murine and human origin, respectively. Collectively, these findings identify HSPA5 in the stress response of rGMCs and implicate regulatory mechanisms that are distinct from those involved in TH inducibility.

Fig 1.

HSPA5 mRNA profiles in metal-stressed rat glomerular mesangial cells (rGMCs). Quantitative reverse transcriptase–polymerase chain reaction measurements of HSPA5 mRNA were conducted in rGMCs exposed to 10 μM actinomycin D (AD), 100 nM thapsigargin (TH) ± AD, 1 and 10 μM HgCl₂ ± AD, and 1 and 10 μM Pb²⁺ acetate ± AD. AD was used as a transcriptional inhibitor. Thapsigargin was used as a positive control for endoplasmic reticulum stress (Baumeister et al 2005) and HSPA5 induction. Total RNA was extracted and processed as described in Materials and Methods. Similar results were seen in 3 separate experiments

Fig 2.

HSPA5 protein expression profiles in metal-stressed rat glomerular mesangial cells (rGMCs). (A) Western blot analysis of HSPA5 protein in rGMCs exposed to 10 μM actinomycin (AD), 1 μM HgCl₂ ± 10 μM AD or 5 μM cycloheximide (Cyc), 300 nM thapsigargin (TH) ± AD or Cyc. (B) Western blot analysis of HSPA5 protein in rGMCs exposed to 10 μM AD, 10 μM HgCl₂ ± 10 μM AD or 5 μM Cyc. (C) Western blot analysis of HSPA5 protein in rGMCs exposed to dimethyl sulfoxide, 1 or 10 μM Pb²⁺ acetate ± 10 μM AD or 5 μM Cyc. Total protein was extracted and processed as described in Materials and Methods. Values represent data from 4 separate experiments, each normalized to Ponceau-S staining and no treatment

Fig 3.

Transcriptional activation profiles in metal-stressed rat glomerular mesangial cells (rGMCs). HSPA5–chloramphenicol acetyltransferase (CAT) constructs were transiently transfected into rGMCs, and CAT activity was measured as described in Materials and Methods. CAT activities were measured in cells challenged with dimethyl sulfoxide, 100 nM TH, 10 μM HgCl₂, or 10 μM Pb²⁺ acetate. Similar results were seen in 3 separate experiments with at least 3 replicate cultures per group

Fig 4.

HSPA5 protein expression profiles in benzo(a)pyrene-stressed rat glomerular mesangial cells (rGMCs). Western blot analysis of HSPA5 protein in rGMCs exposed to 10 μM actinomycin (AD), 100 nM thapsigargin (TH), AD + TD, 3 μM BaP, 3 μM BaP + 10 μM AD, 3 μM BaP + 10 μM HgCl₂, 3 μM BaP + 10 μM Pb²⁺ acetate

Fig 5.

Transcriptional activation profiles in metal-stressed rat glomerular mesangial cells (rGMCs). HSPA5–chloramphenicol acetyltransferase (CAT) constructs were transiently transfected into rGMCs and CAT activity was measured as described in Materials and Methods. CAT activities were measured in cells challenged with dimethyl sulfoxide, 100 nM TH, 3 μM BaP, BaP + 3 μM HgCl₂, or 3 μM BaP + 3 μM Pb²⁺ acetate. Similar results were seen in 3 separate experiments with at least 3 replicate cultures per group

Fig 6.

Immunocytochemical localization of HSPA5 in M15 and HEK293 cells. Images represent data taken from 3 different experiments in which 20 images (∼30 cells/image) per biological triplicate were analyzed for HSPA5 signal, corrected for a no–primary antibody background subtraction, and compared with vehicle control. (A) Representative images from HSPA5; (B, C) quantification

Fig 7.

Schematic representation of the conserved stress-associated putative transcription factor binding sites in the mouse and rat HSPA5 gene promoters. Dark regions represent sequences with high homology between the 2 species. Clear areas represent regions in which no matching was found. Arrows indicate the direction in which the sequence is located. Transcription factor binding sites are depicted to an approximate scale