Makiko Takada

Overexpression of a 60-kDa heat shock protein enhances cytoprotective function of small intestinal epithelial cells.

AIMS With the advancement of small intestinal (double balloon and capsule) endoscopy technology, incidence of small intestinal lesion caused by nonsteroidal anti-inflammatory drugs (NSAIDs) has been known to be high. However, therapy for small intestinal mucosal lesion has not yet been developed. Previous studies have shown that heat shock proteins (HSPs) are involved in cytoprotection mediated by their function as a molecular chaperone. In this study, we examined the effect of HSP60 or HSP70 overexpression on hydrogen peroxide-induced (H2O2) or indomethacin-induced cell damage in the small intestinal epithelial cells. MAIN METHODS cDNA of human HSP60 or HSP70 was transfected to rat small intestinal (IEC-6) cells, and HSP60- or HSP70-overexpressing cells were cloned. IEC-6 cells transfected with vector only were used as control cells. These cells were treated with H2O2 (0-0.14mM) or indomethacin (0-2.5mM). The cell viability was determined by MTT-assay. Cell necrosis was evaluated by LDH-release assay. Further, apoptosis was evaluated by caspases-3/7 activity and TUNEL assay. KEY FINDINGS Cell viability after H2O2 or indomethacin treatment was significantly higher in HSP60-overexpressing cells compared with that in control cells and HSP60-overexpressing cells. Apoptotic cells were also reduced in HSP60-overexpressing. CONCLUSION These results indicate that HSP60 plays an important role in protecting small intestinal mucosal cells from H2O2-induced or indomethacin-induced cell injury. HSP70-overexpressing cells did not show anti-apoptotic ability. SIGNIFICANCE These findings possibly suggest that function of each HSP is different in the small intestine. Therefore, for the therapy of small intestinal mucosal lesion, HSP60-induction therapy could be a new therapeutic strategy.

Target molecules of molecular chaperone (HSP70 family) in injured gastric mucosa in vivo

AIMS Several recent studies, including ours, have indicated the importance of heat shock proteins (HSPs) in cytoprotection against cytotoxic agents and environmental stresses mediated by the chaperone function of HSPs (molecular chaperones). However, the target molecule that is recognized by HSPs in damaged cells currently remains unknown. As HSPs rapidly recognize and bind to degenerated protein in cells, target molecules of HSPs might be key molecules for the initiation and pathogenesis of cellular damage. In the present study, gastric mucosal proteins that specifically bind to the HSP70 family (HSC70) were analyzed using HSC70-affinity chromatography. MAIN METHODS The gastric mucosa was removed from Sprague-Dawley rats after exposure to water immersion-stress for 0, 1, 3 or 5 h. Soluble fractions of each gastric mucosa were applied to the HSC70-affinity column separately. After washing off non-specific binding proteins, specific binding proteins were eluted by ATP-containing buffer. Binding proteins were analyzed by SDS-polyacrylamide gel electrophoresis. In addition, the amino acid sequence of purified proteins was also analyzed. KEY FINDINGS Specific HSC70-binding proteins with a molecular weight of 200-kDa and 45-kDa were eluted from an affinity column when gastric mucosal homogenate of 1-h stress exposure was applied. The amino acid sequencing showed that these binding proteins were cytoskeletal myosin (heavy chain) and actin, respectively. SIGNIFICANCE During the pathogenesis of stress-induced gastric mucosal damage, structurally degenerated cytoskeletal myosin (heavy chain) and actin may be key or initiation molecules which structural changes were firstly recognized by molecular chaperone.

Evidence for Enhanced Cytoprotective Function of HSP90-Overexpressing Small Intestinal Epithelial Cells

BackgroundIn the clinical field, increasing incidence of small intestinal ulcers associated with nonsteroidal anti-inflammatory drugs (NSAIDs) has become a topic with the advances of capsule endoscopy and balloon enteroscopy technology for the detection of small intestinal lesions. However, the pathogenesis of NSAID-induced mucosal damage, defensive mechanism of intestinal epithelial cells, and therapy for small intestinal mucosal lesion have not been fully understood. Heat shock proteins (HSPs) are involved in cytoprotection mediated by their function as a molecular chaperone. Since the function of HSP90 in the intestinal epithelial cells has not been well investigated, we examined the cytoprotective ability of HSP90-overexpressing small intestinal epithelial cells against hydrogen peroxide-induced or indomethacin-induced cell damage.MethodscDNA of human HSP90 gene was transfected to rat small intestinal epithelial cells (IEC-6 cells), and HSP90-overexpressing cells (IEC-6-90 cells) were selected and cloned. Anti-necrotic abilities and anti-apoptotic abilities of IEC-6-90 cells were compared with IEC-6-mock cells (transfected with vector alone). To examine the specific contribution of HSP90 on cytoprotection of IEC-6-90 cells, cytoprotective ability of IEC-6-90 cells was analyzed with or without pretreatment with functional inhibitor of HSP90, geldanamycine analog, followed by hydrogen peroxide-challenge or indomethacin-challenge.ResultsHydrogen peroxide-induced or indomethacin-induced cell necrosis and apoptosis were significantly suppressed in IEC-6-90 cells. The cytoprotective ability of IEC-6-90 cells was suppressed by HSP90 inhibitor.ConclusionsOur results suggest that HSP90 might play an important role in protecting small intestinal epithelial cells from hydrogen peroxide-induced or indomethacin-induced cell injury in vitro, and raised the possibility of protection of small intestinal epithelial cells by manipulation of HSP90 expression.

Specific induction of a 72-kDa heat shock protein protects esophageal mucosa from reflux esophagitis

AIMS The aim of this study is to investigate the expression and cytoprotective function of a 72-kDa heat shock protein (HSP72) using a reflux esophagitis model in rats. MAIN METHODS Expression of HSP60, HSP72, and HSP90 in rat esophageal mucosa was evaluated by Western blot analysis before and after hyperthermia (42.5 degrees C, 20 min). Rats received the operation to produce reflux esophagitis with or without pretreatment with hyperthermia to induce HSPs. The esophageal mucosal damage was evaluated 12 h after the operation. KEY FINDINGS Expression of HSP72 was significantly increased by hyperthermia in rat esophageal mucosa. Reflux esophagitis was dramatically prevented when HSP72 was preinduced by hyperthermia. Furthermore, activation of TNF-alpha and IL-1beta in esophageal mucosa was also suppressed. SIGNIFICANCE These results suggested that hyperthermia protects the esophageal mucosa in reflux esophagitis model by inducing HSP72 and suppressing proinflammatory cytokine activation. These findings might suggest that HSP-inducing therapy could be a novel and unique therapy for reflux esophagitis.

Correlation of Heat Shock Protein Expression to Gender Difference in Development of Stress-Induced Gastric Mucosal Injury in Rats

Recent studies have indicated that heat shock proteins (HSPs), which function as molecular chaperones, play important roles in cellular responses to stress-related events. However, the gender difference in the expression of HSP in the gastric mucosa remains unclear. In order to understand the mechanism of gender difference in the prevalence or severity of gastric mucosal lesions, the expression level of HSP and the correlation of estrogen to HSP induction in the gastric mucosa were evaluated in this study. The basal expression levels of HSP60 and HSP90 in the gastric mucosa were significantly higher in females than those in males. The gastric ulcer index was significantly higher in male rats compared to female rats observed after 12 h water immersion stress exposure. At this time point, the expression levels of HSP60 and HSP90 in the gastric mucosa were significantly higher in females than those in males. An estrogen-treatment significantly induced the expression of HSP60, HSP70 and HSP90 in the gastric mucosa. Inversely, an ovariectomy dramatically reduced the expression of HSP60, HSP70 and HSP90 in the gastric mucosa. Our results suggested that estrogen might play an important role in gastric mucosal protection with the induction of gastric mucosal HSPs.