Small heat shock protein speciation: novel non-canonical 44 kDa HspB5-related protein species in rat and human tissues.

When analyzing small stress proteins of rat and human tissues by electrophoretic methods followed by western blotting, and using the anti-HspB1/anti-HspB5 antibody clone 8A7, we unexpectedly found a protein with a molecular mass of ~44 kDa. On two-dimensional gels, this protein resolved into four distinct species. Electrophoretic and immunological evidence suggests that this 44 kDa protein is a derivative of HspB5, most likely a covalently linked HspB5 dimer. This HspB5-like 44 kDa protein (HspB5L-P44) is particularly abundant in rat heart, brain, and renal cortex and glomeruli. HspB5L-P44 was also found in human brains, including those from patients with Alexander disease, a condition distinguished by cerebral accumulation of HspB5. Gray matter of such a patient contained an elevated amount of HspB5L-P44. A spatial model of structurally ordered dimeric HspB5 α-crystallin domains reveals the exposed and adjacent position of the two peptide segments homologous to the HspB1-derived 8A7 antigen determinant peptide (epitope). This explains the observed extraordinary high avidity of the 8A7 antibody towards HspB5L-P44, as opposed to commonly used HspB5-specific antibodies which recognize other epitopes. This scenario also explains the remarkable fact that no previous study reported the existence of HspB5L-P44 species. Exposure of rat endothelial cells to UV light, an oxidative stress condition, temporarily increased HspB5L-P44, suggesting physiological regulation of the dimerization. The existence of HspB5L-P44 supports the protein speciation discourse and fits to the concept of the protein code, according to which the expression of a given gene is reflected only by the complete set of the derived protein species.

p38 MAPK/HSP25 signaling mediates cadmium-induced contraction of mesangial cells and renal glomeruli.

The environmental pollutant cadmium affects human health, with the kidney being a primary target. In addition to proximal tubules, glomeruli and their contractile mesangial cells have also been identified as targets of cadmium nephrotoxicity. Glomerular contraction is thought to contribute to reduced glomerular filtration, a characteristic of cadmium nephrotoxicity. Because p38 MAPK/HSP25 signaling has been implicated in smooth muscle contraction, we examined its role in cadmium-induced contraction of mesangial cells. We report that exposure of mesangial cells to cadmium resulted in 1) cell contraction, 2) activation of MAP kinases, 3) increased HSP25 phosphorylation coincident with p38 MAP kinase activation, 4) sequential phosphorylation of the two phosphorylation sites of mouse HSP25 with Ser15 being phosphorylated before Ser86, 5) reduction of oligomeric size of HSP25, and 6) association of HSP25 with microfilaments. Exposure of isolated rat glomeruli to cadmium also resulted in contraction and increased HSP25 phosphorylation. The cadmium-induced responses were inhibited by the specific p38 MAP kinase inhibitor SB-203580, and cadmium-induced phosphorylation of HSP25 was inhibited by expression of a dominant-negative p38 MAP kinase mutant. These findings tentatively suggest that cadmium-induced nephrotoxicity results, in part, from glomerular contraction due to p38 MAP kinase/HSP25 signaling-dependent contraction of mesangial cells. With regard to the cellular action of HSP25, these data support a change in paradigm: in addition to its well-established cytoprotective function, HSP25 may also be involved in processes that ultimately lead to adverse effects, as is observed in the response of mesangial cells to cadmium.

HSP27 regulates fibroblast adhesion, motility, and matrix contraction.

Heat shock protein 27 (HSP27) modulates actin-dependent cell functions in several systems. We hypothesized that HSP27 modulates wound contraction. Stably transfected fibroblast cell lines that overexpress HSP27 (SS12) or underexpress HSP27 (AS10) were established, and cell behaviors related to wound contraction were examined. First, fibroblast-populated collagen lattice (FPCL) contraction was examined because it has been studied as a wound-healing model. In floating FPCL contraction assays, SS12 cells caused increased contraction, whereas AS10 cells caused reduced contraction. Because floating matrix contraction is thought to be mediated by the tractional force of the cells, cell behaviors related to tractional force were examined. In collagen matrix, SS12 cells elongated faster and to a greater extent and contained longer stress fibers than control cells, whereas AS10 cells were slower to elongate than control cells. SS12 cells attached to the dishes more efficiently than the control, whereas AS10 cells attached less efficiently. Migration of SS12 cells on collagen-coated dishes was also enhanced, although AS10 cells did not differ from the control cells. In summary, HSP27 regulates fibroblast adhesion, elongation, and migration and the contraction of the floating matrix in a manner dependent on the level of its expression.

MAP kinase pathways involving hsp27 regulate fibroblast-mediated wound contraction.

BACKGROUND: Heat shock protein 27 (hsp27) has been shown to modulate actin arrays in a manner dependent on its phosphorylation status. Hsp27 is phosphorylated by mitogen-activated protein kinase-activated protein kinase 2/3, which is regulated by mitogen-activated protein (MAP) kinases. We hypothesize that hsp27 phosphorylation modulates wound contraction. MATERIALS AND METHODS: In these studies, a specific p38 MAP kinase inhibitor, SB203580, and a specific MAPK/extracellular signal-regulated kinase kinase 1,2 inhibitor, PD98059, were used to inhibit kinase activity. The effect of MAP kinase inhibitors was tested using a tissue culture model, the fibroblast-populated collagen lattice (FPCL) contraction assay, and a rat full-thickness skin defect model of wound healing. Hsp27 phosphorylation status was determined by isoelectric focus and Western blot analysis. RESULTS: We show here that hsp27 phosphorylation correlates with FPCL contraction and with contraction in vivo. In the tissue culture model, each inhibitor reduced FPCL contraction and hsp27 phosphorylation. Hsp27 phosphorylation correlated with both p38 and ERK1, 2 activation. Hsp27 was highly phosphorylated in the wound edge during wound healing in a rat in vivo model. The phosphorylation status was highest in the granulation tissue. Treatment with both kinase inhibitors significantly delayed wound contraction in vivo, which correlated with inhibition of hsp27 phosphorylation. CONCLUSIONS: This study demonstrates that ERK and p38 kinase cascades play important roles in wound contraction. Additionally, these data implicate hsp27 as being a key molecule in modulating the effects of these kinases.