Protective role of HSF1 and HSP70 against gastrointestinal diseases

Abstract

Purpose: It is well known that heat shock proteins (HSPs) are induced by various stressors in order to confer protection against such stressors. Since stressor-induced tissue damage is involved in various diseases, especially gastrointestinal diseases, it has been thought that HSP-inducers are therapeutically beneficial for these diseases. Indirect lines of evidence suggest that HSPs provide a major protective mechanism against irritant-induced gastric lesions. However, no direct evidence exists to support this notion. On the other hand, inflammatory bowel disease (IBD) involves infiltration of leukocytes into intestinal tissue, resulting in intestinal damage. Pro-inflammatory cytokines and cell adhesion molecules (CAMs) play important roles in this infiltration of leukocytes. The roles of heat shock factor 1 (HSF1, a transcription factor for hsp genes) and heat shock proteins (HSPs) in development of IBD are unclear. In this paper, we reviewed our recent work on the role of HSPs in pathogenesis of gastric lesions and IBD by use of HSF1-null mice and transgenic mice expressing Hsp70.

Conclusion: This study provides the first genetic evidence that HSF1 and Hsp70 play a role in protecting against both irritant-induced gastric lesions and IBD-related colitis. The aggravation of irritant-induced gastric lesions in HSF1-null mice is due to their inability to up-regulate Hsp70, leading to apoptosis. On the other hand, this protective role of Hsp70 against colitis seems to involve various mechanisms, such as suppression of expression of pro-inflammatory cytokines and CAMs, and cell death.

• HSF1
• Hsp70
• gastrointestinal diseases
• IBD
• pro-inflammatory cytokines
• CAMs

Introduction

The balance between aggressive and defensive factors determines development of gastric lesions. The gastric mucosa is challenged by a variety of both endogenous and exogenous irritants (aggressive factors), including ethanol, gastric acid, pepsin, non-steroidal anti-inflammatory drugs (NSAIDs) and Helicobacter pylori. These irritants damage the mucosal cells, inducing cell death which leads to the formation of gastric lesions [1]. In order to protect the gastric mucosa, a complex defence system, which includes the production of surface mucus and bicarbonate and the regulation of gastric mucosal blood flow (GMBF), has evolved. Prostaglandins (PGs), in particular PGE₂, enhance these protective mechanisms, and are therefore thought to comprise a major gastric mucosal defensive factor [2].

Recently, heat shock proteins (HSPs) have also attracted considerable attention as another major defensive factor. When cells are exposed to stressors, a number of so-called stress proteins are induced, in order to confer protection against such stressors. HSPs are representative of these stress proteins, and their cellular up-regulation, especially that of Hsp70, provides resistance as they re-fold or degrade denatured proteins produced by the stressors [3]. It has been reported not only that various gastric irritants, including ethanol, up-regulate HSPs, but also that artificial up-regulation of HSPs confers resistance to these irritants in cultured gastric mucosal cells [3]. Although these findings strongly indicate that HSPs are protective, very little direct evidence exists, and to date no in vivo study has been conducted to demonstrate that inhibition of HSPs results in a phenotype susceptible to irritant-induced gastric lesions.

Interestingly, geranylgeranylacetone (GGA), a leading anti-ulcer drug on the Japanese market, has been reported to be a non-toxic HSP-inducer, up-regulating various HSPs not only in cultured gastric mucosal cells at concentrations that do not affect cell viability but also in various tissues, including the gastric mucosa in vivo [4],[5]. We have previously reported that pre-induction of HSPs by GGA protects cultured gastric mucosal cells from cell death induced by various irritants [6–9]. These previous results suggest that the anti-ulcer effect of GGA is due to its HSP-inducing activity. However, because GGA mediates various other gastro-protective mechanisms [10],[11], it remains unclear whether up-regulation of HSPs represents GGA's major mode of anti-ulcer activity. The up-regulation of HSPs by various stressors is regulated at the transcription level by a consensus cis-element (heat shock element (HSE)) and a transcription factor (heat shock factor 1 (HSF1)), that specifically binds to HSE located on the upstream region of hsp genes [12].

Inflammatory bowel disease (IBD), Crohn's disease (CD) and ulcerative colitis (UC), have become substantial health problems with an actual prevalence of 200–500 per 1 00 000 people in western countries, which almost doubles every 10 years [13]. Although the etiology of IBD is not yet fully understood, recent studies suggest that IBD involves chronic inflammatory disorders in the intestine due to ‘a vicious cycle’. Infiltration into intestinal tissues cause intestinal mucosal damage induced by reactive oxygen species (ROS) that are released from the activated leukocytes, and this intestinal mucosal damage further stimulates the infiltration of leukocytes [14]. To understand the molecular mechanism underlying the pathogenesis of IBD and to develop new types of clinical drugs for IBD, identification of endogenous factors that positively or negatively affect the development of IBD is important. For this purpose, various experimental animal colitis models, in particular the dextran sulphate sodium (DSS)-induced colitis models have been used [15]. Pro-inflammatory cytokines and cell adhesion molecules (CAMs) play an important role in the activation and infiltration of leukocytes that is associated with IBD [16],[17].

HSPs and HSF1 also have attracted considerable attention as candidates for endogenous factors that affect the development of IBD because some HSPs, including Hsp70, were reported to be over-expressed in the intestinal tissues of IBD patients and in an animal model of IBD [18]. HSF1 negatively regulates the expression of tnf-α and il-1β genes [19]. These previous results suggest that HSPs and HSF1 have negative roles on the development of IBD, however, there is no direct evidence (such as genetic evidence) to support this idea. Furthermore, there are some data that suggest positive roles for HSPs and HSF1 in the development of IBD [20]. Therefore, the effects of genetic alteration of HSPs and HSF1 on the development of colitis in animal models of IBD should be examined in order to understand the exact role (positive or negative) of HSPs and HSF1 in IBD.

In this review, we summarised our recent work using HSF1-null mice and transgenic mice expressing Hsp70 to obtain direct genetic evidence for the contribution of HSPs to the protection of the gastric mucosa and to examine the role of HSF1 and HSPs in the pathogenesis of DSS-induced colitis.

Results

The role of HSF1 and HSPs in gastric ulcerogenic response

We examined the role of HSF1 in gastric ulcerogenic response by use of HSF1-null mice [21]. As shown in Figure 1A, intragastric administration of 40% ethanol resulted in significant gastric lesions in HSF1-null mice but not in wild-type mice. These results show that HSF1 plays an important role in protecting the gastric mucosa from ethanol-induced lesions. Similar results were obtained with hydrochloric acid-induced gastric lesions (Figure 1B). Given that HSF1 up-regulates the expression of HSPs, we examined the effect of ethanol administration on the expression of HSPs in the gastric mucosa of HSF1-null mice and wild-type mice. Ethanol administration up-regulated the production of only Hsp70, a response that was dependent on the function of HSF1 (Figure 1C). Based on these results, we subsequently focused on Hsp70.

Figure 1. Production of gastric lesions and expression of HSPs in wild-type and HSF1-null mice. Wild-type (WT) and HSF1-null mice (HSF1-KO) were orally administered the indicated doses of ethanol (A, C) or hydrochloric acid (B). After 4 h, the stomach was removed and scored for haemorrhagic damage. Values are mean ± SEM (n = 4–6). *P < 0.05 (A, B). After 4 h, the gastric mucosa was removed, and protein extracts were prepared and analysed by immunoblotting with an antibody against Hsp25, Hsp60, Hsp70, Hsp90, or actin (C). This figures was published previously and is reprinted here with permission of the journal [21].

In order to investigate the mechanism governing the severity of production of ethanol-stimulated gastric lesions in HSF1-null mice, we compared the level of apoptosis. In wild-type mice, an increase in TUNEL-positive (apoptotic) cells was observed following the administration of 100% but not 40% ethanol, whereas a clear increase in TUNEL-positive cells was observed with 40% ethanol administration in the HSF1-null mice [21]. These results show that induction of apoptosis by ethanol is enhanced in HSF1-null mice compared to wild-type mice; in other words, HSF1 protects gastric mucosal cells from ethanol-induced apoptosis.

Another group also suggested the protective role of HSPs (Hsp27) against irritant-induced gastric lesions. Hsp27 also has cytoprotective effect. Ebert et al. reported that transgenic mice expressing Hsp27 showed resistant phenotype to indometacin-induced gastric lesions. They also showed that overexpression of Hsp27 did not affect the expression of COX-1 and COX-2, suggesting that expression of Hsp27 protects gastric mucosa against NSAID-induced lesions through cytoprotection rather than modulation of inhibition of COX by NSAIDs [22].

In order to evaluate the contribution of the HSP-inducing activity of GGA to its anti-ulcer activity, we investigated the effect of GGA in HSF1-null mice. First, we examined the effect of GGA and/or ethanol on gastric mucosal Hsp70 expression in wild-type mice, revealing a potent expression induced by ethanol, and a lower level of expression in response to GGA (Figure 2A). Interestingly, pre-administration of GGA enhanced the ethanol-dependent Hsp70 response (Figure 2A). We confirmed that administration of GGA and/or 40% ethanol did not induce Hsp70 (Figure 2B). Figure 2C shows the effect of pre-administration of GGA on ethanol-produced gastric lesions in wild-type and HSF1-null mice. In order to obtain similar levels of gastric lesions, 100% and 40% ethanol administration were administered to wild-type and HSF1-null mice, respectively. In fact, 40% ethanol administration in HSF1-null mice caused the comparable lesion score as 100% ethanol administration in wild-type mice (Figure 2C). Pre-administration of GGA significantly suppressed the ethanol-dependent production of gastric lesions in wild-type mice (Figure 2C). In contrast, no significant effect was recorded in the HSF1-null mice (Figure 2C). This result shows that HSF1 is required for the efficacy of the anti-ulcer activity of GGA against ethanol. Overall, the results in Figure 2 suggest that the loss of the protective effect of GGA in HSF1-null mice is due to the lack of expression of HSPs (such as Hsp70); in other words, the HSP-inducing activity of GGA contributes to its anti-ulcer activity.

Figure 2. Effect of ethanol and/or GGA on expression of Hsp70 and production of gastric lesions. Wild-type (WT) (A, C) and HSF1-null (HSF1-KO) (B, C) mice were orally pre-administered 200 mg/kg GGA (10 mL/kg as emulsion with 5% gum arabic), 1 h after which they were orally administered with the indicated doses of ethanol. After 4 h, sections of gastric tissues were prepared and subjected to histological examination (H&E) and immunohistochemical analysis with an antibody against Hsp70 (A, B). After 4 h, the stomach was removed and scored for haemorrhagic damage. Values are mean ± SEM (n = 3–6). *P < 0.05. n.s., not significant (C). This figure was published previously and is reprinted here with permission of the journal [21].

The role of HSF1 and HSPs in IBD-related colitis

We examined the role of HSF1 and Hsp70 in DSS-induced colitis by use of HSF1-null mice and transgenic mice expressing Hsp70 [23]. The severity of DSS-induced colitis can be monitored by various indexes, such as body weight, Disease Activity Index (DAI), length of colon and MPO activity. Administration of 3% DSS caused a mild increase in the DAI but did not affect the body weight of the wild-type mice. In contrast, administration of 3% DSS resulted in a higher DAI score and loss of body weight in HSF1-null mice [23]. DSS-induced colon shortening, used as a morphometric measure for the degree of inflammation, was more severe in HSF1-null mice than in the wild-type mice [23]. Colonic MPO activity, an indicator of infiltration of leukocytes, was much higher in DSS-administered HSF1-null mice than the wild-type mice [23]. The results show that HSF1-null mice are more sensitive to DSS-induced colitis than their respective wild-type mice. We monitored expression of hsp mRNAs in colonic tissues of DSS-administered and untreated HSF1-null mice and wild-type mice by real-time RT-PCR. The expression of hsp70 but not hsp25 and hsp60 mRNAs was significantly lower in the DSS-treated HSF1-null mice than in the wild-type mice [23]. Development of DSS-induced colitis was compared in transgenic mice expressing Hsp70 and their respective wild-type mice. DSS-dependent increase in DAI was clearly suppressed in transgenic mice expressing Hsp70 compared to the wild-type mice [23]. All of the other indexes of colitis that were tested (colon length and colonic MPO activity) showed that transgenic mice expressing Hsp70 are more resistant than the wild-type mice to DSS-induced colitis [23]. The results suggest that Hsp70 expression somehow suppresses DSS-induced colitis.

Another group addressed this issue by use of GGA and mice. Ohkawara et al. reported that oral administration of GGA (300–500 mg/kg) suppressed DSS-induced increase in DAI, colon shortening, increase in colonic MPO activity and colonic mucosal damage. They also showed that administration of GGA lowered the colonic level of pro-inflammatory cytokines. Furthermore, they showed that GGA up-regulated the expression of Hsp70 and Hsp40 but not other HSPs in the colons [24]. They also examined the effect of GGA on trinitrobenzene sulphonic acid (TNBS)-induced colitis, another animal model of IBD. Oral administration of GGA (300 mg/kg) suppressed TNBS-induced decrease in body weight, increase in DAI, increase in colonic MPO activity and colonic mucosal damage. They also showed that the survival rate of mice treated with TNBS significantly increased by GGA administration [25]. These results support the idea that HSPs are protective against IBD-related colitis and suggest that non-toxic inducers of HSP expression are therapeutically beneficial for IBD.

Tao et al. reported that Hsp70-null mice are susceptible to azoxymethane (AOM)/DSS-induced colitis (such as mucosal damage and inflammation). They also showed that AOM/DSS-induced colonic tumours were promoted in Hsp70-null mice, compared to wild-type mice [26].

Mechanism for protective role of Hsp70 against colitis

In order to understand the mechanism governing the decreased susceptibility of transgenic mice expressing Hsp70 to DSS-induced colitis, we compared the mRNA expression of various inflammation-related proteins in the colonic tissues [23]. As shown in Figure 3A, the mRNA expression of tnf-α, il-1β and il-6 in colonic tissues was significantly lower in DSS-administered transgenic mice expressing Hsp70 than in the wild-type mice, suggesting that Hsp70 negatively regulates the expression of the selected pro-inflammatory cytokines under inflammatory conditions. To test this idea in vitro, we compared the LPS-stimulated production of the pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) in peritoneal macrophages prepared from the transgenic mice and their wild-type counterparts. LPS stimulated the production of all of these pro-inflammatory cytokines and the levels of not only TNF-α but also of IL-1β and IL-6 were much lower in the medium of the LPS-treated macrophages prepared from transgenic mice expressing Hsp70 than from wild-type mice [23]. These results suggest that expression of Hsp70 may suppress the production of these pro-inflammatory cytokines under inflammatory conditions.

Figure 3. The mRNA expression of various genes and levels of cell death in colonic mucosa. Transgenic mice expressing Hsp70 (Hsp70 Tg) and wild-type mice (WT) were treated with or without 3% DSS for 7 days (A, B). Relative mRNA expression of each gene in colonic tissues was monitored and expressed. Values are mean ± SEM (n = 3–6). **P < 0.01; *P < 0.05 (A). Sections of colonic tissues were prepared and subjected to TUNEL assay and DAPI staining (B). This figure was published previously and is reprinted here with permission of the journal [23].

We compared mRNA expression of vcam-1, icam-1 and madcam-1 in the colonic tissues of transgenic mice expressing Hsp70 and wild-type mice. The mRNA expression of these CAMs was much lower in the DSS-administered transgenic mice than the wild-type mice (Figure 3A), suggesting that expression of CAMs is negatively regulated by Hsp70 under inflammatory conditions. To test this idea in vitro, we examined the effect of siRNA specific for HSF1 or Hsp70 on the LPS-induced mRNA expression of the CAMs in bEnd.3 (mouse brain endothelioma) cells. Transfection with HSF1 siRNA up-regulated the mRNA expression of vcam-1 and icam-1 but down-regulated that of madcam-1 in the presence of LPS. Transfection of the cells with siRNA specific for Hsp70 did not significantly up-regulate the mRNA expression of the CAMs in the presence of LPS, suggesting that, at least in vitro, Hsp70 does not negatively regulate the mRNA expression of these CAMs under inflammatory conditions [23].

We compared the level of cell death in the colonic mucosa of DSS-administered transgenic mice expressing Hsp70 and the respective wild-type mice by use of the TUNEL assay. Less TUNEL-positive cells were observed in the colonic mucosa of DSS-administered transgenic mice expressing Hsp70 than the wild-type mice (Figure 3B). The results suggest that ROS-induced cell death associated with DSS-induced colitis is suppressed in transgenic mice expressing Hsp70, respectively.

To test the role of Hsp70 in ROS-induced cell death in vitro, we examined the effect of siRNA specific for Hsp70 on cell death induced by menadione, a superoxide anion (a representative ROS) releasing drug, in a colonic cancer cell line (HCT-15). Transfection of cells with siRNA for Hsp70 clearly stimulated cell death induced by menadione [23]. The results suggest that Hsp70 protects colonic cells from ROS-induced cell death and that this effect may be involved in the improved resistance to DSS-induced colitis that is observed in transgenic mice expressing Hsp70.

Discussion

A number of previous observations have suggested that HSPs and their up-regulation by gastric irritants play an important role in protecting the gastric mucosa against lesion development [4],[6–9],[27]. We found that HSF1-null mice are more susceptible to irritant-induced gastric lesions, providing direct genetic evidence for the significance of HSPs in ameliorating the outcome of irritant-induced gastric insults (Figure 4A).

Figure 4. The proposed mechanism for protective role of HSF1 and Hsp70 against gastric ulcer (A) and IBD (B).

GGA has attracted considerable attention as an HSP-inducer, largely due to its clinical value as an anti-ulcer drug and because it can induce HSPs without affecting cell viability [4]. GGA has been suggested to play a protective role through HSP-induction in a variety of disease states [5],[28]. However, no previous reports have shown that the HSP-inducing activity of GGA contributes to these clinically beneficial outcomes, including its anti-ulcer effects. Using immunohistochemical analysis, we have demonstrated that oral administration of GGA alone up-regulates gastric mucosal Hsp70, and that pre-administration of GGA stimulates the ethanol-induced up-regulation of Hsp70. Furthermore, we have revealed that pre-administration of GGA suppresses gastric lesions in wild-type mice but not in HSF1-null mice [21]. These results argue strongly in favour of the HSP-inducing activity of GGA contributing to its anti-ulcer effects, providing the first direct genetic link between the pharmacological behaviour of the drug and the resultant clinical outcome.

Because some HSPs have been reported to be over-expressed in the intestinal tissues of IBD patients [18],[29],[30], HSF1 and HSPs are thought to be involved in the pathogenesis of IBD. We have gathered evidence that HSF1 and HSPs have negative roles in the development of IBD (protective roles against IBD) by demonstrating the sensitive phenotype of HSF1-null mice and the resistant phenotype of transgenic mice expressing Hsp70 (or HSF1) against DSS-induced colitis, an animal model for IBD [23]. Furthermore, we have examined the molecular mechanisms governing the susceptibility of HSF1-null mice and resistance of transgenic mice expressing Hsp70 to DSS-induced colitis, focusing on the expression of pro-inflammatory cytokines and CAMs and ROS-induced cell death both in vivo and in vitro (Figure 4B).

Pro-inflammatory cytokines, in particular TNF-α, positively contribute to the progression of IBD [31],[32]. DSS-induced mRNA expression of various pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) in colonic tissues was inhibited in transgenic mice expressing Hsp70 [23]. We consider that this inhibition of cytokine mRNA expression is responsible for the DSS-induced colitis phenotypes exhibited by the mice. The LPS-induced production of not only TNF-α but also IL-1β and IL-6 was inhibited in peritoneal macrophages prepared from transgenic mice expressing Hsp70 [23], suggesting that Hsp70 suppresses the production of these pro-inflammatory cytokines under inflammatory conditions (Figure 4B). We speculate that this suppression is mediated by Hsp70-dependent inhibition of nuclear factor kappa B (NF-κB), which plays an important role in the induction of inflammation. It is known that NF-κB positively regulates expression of pro-inflammatory cytokines including TNF-α, IL-1β and IL-6. Furthermore, it is also known that up-regulation of Hsp70 expression by heat shock inhibits the inflammatory stimuli-dependent activation of NF-κB through various mechanisms [33–39].

CAMs also positively contribute to the progression of IBD through recruitment of blood circulating leukocytes into inflamed intestinal tissues [17]. DSS-administration induced mRNA expression of CAMs that are mainly expressed on vascular endothelial cells and of those on leukocytes. However, HSF1 deficiency only affected mRNA expression of CAMs on vascular endothelial cells [23], suggesting that these CAMs rather than those on leukocytes contribute to the greater sensitivity of HSF1 null mice, relative to their wild-type controls, to DSS-induced colitis. The DSS-induced mRNA expression of ICAM-1, VCAM-1 and MAdCAM-1 was suppressed in transgenic mice expressing Hsp70 [23], and this may be involved in conferring resistance to DSS-induced colitis (Figure 4B).

Colonic mucosal cell death induced by ROS released from activated leukocytes is thought to be directly responsible for the pathogenesis of human IBD [14]. Analysis using the TUNEL assay revealed that cell death in colonic mucosa was inhibited in transgenic mice expressing Hsp70, respectively. This correlates with other parameters for DSS-induced colitis, however, it was not clear whether these alterations to cell death cause or result from the progression of DSS-induced colitis. Given that transfection with siRNA for Hsp70 stimulated ROS-induced cell death in vitro [23], this result suggests that Hsp70 protects colonic mucosal cells from ROS-induced cell death, which seems to contribute to the lower level of cell death seen in the colonic mucosa of DSS-administered transgenic mice expressing Hsp70 (Figure 4B).

The results of this study suggest that non-toxic inducers of HSP expression are therapeutically beneficial for IBD. Supporting this notion, GGA suppresses both DSS- and TNBS-induced colitis [24],[25]. However, the ability of GGA to induce HSP expression is not strong, which may explain its relatively weak effect on these types of colitis [24],[25]. Therefore, we propose that more potent non-toxic HSP inducers would be therapeutically beneficial for IBD.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.