Regulation of heat shock proteins in the intestine by dietary fibers

概要

Doctoral Thesis

Regulation of heat shock proteins in the intestine
by dietary fibers

[Summary]

Adesina Precious Adedayo

Graduate School of Biosphere Science
Hiroshima University

September 2021

1. Introduction
Disrupted intestinal barrier can lead to entry of noxious luminal contents into the circulation,
triggering the immune response and causing chronic inflammatory diseases such as chronic
kidney disease, cardiovascular disease, and diabetes. This highlights the importance of
maintaining the intestinal epithelial integrity and human health.
Studies have identified the crucial role of heat shock proteins (HSP) in maintaining and
protecting the integrity of intestinal epithelial cells. HSPs restore damaged intracellular
proteins to protect the function, barrier integrity, and survivability of epithelial cells under
stressful conditions. For instance, the induction of HSP25 and HSP70 expression was shown
to promote cytoprotection in intestinal epithelial cells and linked to the suppression of
experimental colitis in mice. Thus, the regulation of HSP expression by dietary components
could be a potential therapeutic strategy to promote intestinal health.
Several studies have highlighted that dietary fibers (DFs) promote intestinal homeostasis.
Intestinal microbiota ferments DFs to generate different metabolites including short chain fatty
acids (SCFAs) such as acetate, propionate, and butyrate. Previous studies in our laboratory
demonstrated that fermentable DFs, guar gum (GG) and partially hydrolyzed GG (PHGG),
reduced intestinal inflammation and barrier defects in a murine model of colitis, suggesting
that the microbial metabolites such as SCFAs have a crucial role in maintaining the intestinal
homeostasis. Whereas intact GG is shown to upregulate the suppressor of cytokine signaling1
(SOCS1) through activation of toll-like receptor (TLR)-2 and dectin-1 signaling pathways to
reduce inflammation in the small intestine of mice.
Since both HSP25 and HSP70 are attributed to intestinal cytoprotection, the purpose of this
study was to examine the effect of GG and PHGG on intestinal HSP25 and HSP70 expression
in mice. The study also highlights the importance DF fermentation by microbiota to confer the

physiological benefits of DFs in the colon. Furthermore, the regulation of HSP70 expression
by SCFA was examined using human intestinal Caco-2 cells in in vitro studies.
2. Regulation of intestinal HSP25 and HSP70 expression by GG in mice
The study examined if GG had a promotive effect on HSP25 and HSP70 expression in the
intestine of mice. The results showed that feeding GG increased the HSP70 protein expression
in epithelial cells of both the small intestine and colon but did not affect HSP25. These findings
suggest that the structure of intact GG directly stimulate the epithelial cells to increase the
HSP70 in the small intestines. Whereas the microbial metabolites of GG such as SCFAs have
a role in the HSP70 expression in the colon. Feeding GG fiber diet increased SCFA production,
such as acetate, propionate, and n-butyrate.
3. The effects of GG in comparison with PHGG on HSP regulation in mice
PHGG is produced through the controlled enzymatic hydrolysis of GG fiber and has low
viscosity, unlike GG. To determine whether the viscosity of GG plays a role in the increased
HSP70 expression in mouse intestine, PHGG was administered to the mice. Like GG, feeding
PHGG increased the HSP70 expression in both small intestine and colon of mice. However,
no effect on HSP25 expression was observed. Supplemental GG and PHGG fibers increased
the cecal acetate, propionate, and n-butyrate in mice. These results suggest that the HSP70
expression by GG and PHGG is independent of their difference in viscosities. Whereas the
increased SCFAs production may be responsible for the upregulation of colonic HSP70 in mice
fed GG and PHGG fibers. To clarify if the bacterial generated SCFAs are important in the
HSP70 expression, the HSP70 expression in the colon of germ-free (GF) mice was examined
in the next study.
4. Influence of microbiota activity on colonic HSP70 expression
This study utilized specific pathogen free (SPF) and GF mice and the HSP70 expression in the

colon and cecal SCFA profile was determined. As a result, HSP70 expression in the colon of
SPF mice was significantly higher compared to GF mice. Acetate, propionate, and butyrate
were highly generated in the cecum of SPF mice, whereas their production was barely observed
in GF mice. Considering these two observations, we suggest that colonic bacteria activity:
namely fermentation and generation of SCFAs is closely involved in the HSP70 expression in
the colon of SPF mice. This result supports the notion that HSP70 expression in the colon is
dependent on microbial signals or metabolites. Hence, the next study investigated the effect of
SCFAs on HSP70 regulation and the underlying mechanisms.
5. Regulation of HSP70 expression in Caco-2 Cells by SCFA
In this section, in vitro studies were carried out to elucidate roles of SCFAs on HSP70
expression using human intestinal Caco-2 cells. Propionate, butyrate but not acetate, increased
the HSP70 protein expression in Caco-2 cells. Phosphorylation (activation) of heat shock
factor1 (pHSF1), a well-known transcriptional factor of HSP70, was also increased by
propionate and butyrate, but not acetate. The qRT-PCR analysis demonstrated the increase in
Hspa1a (HSP70) mRNA levels by propionate and butyrate in a dose-dependent manner.
Propionate and butyrate increased Hspa1a promoter activity in a dose-dependent manner
indicating that the HSP70 expression induced by propionate and butyrate occurs at
transcriptional level. The pharmacological inhibition of MEK and mTOR kinases
downregulated HSP70 expression by propionate and butyrate. The result also reveals that at
least these 2 kinases are important in the activation of HSF1. Propionate and butyrate can
modulate cellular functions via their ability to inhibit histone deacetylases (HDAC).
Trichostatin A (TSA), a well-known HDAC inhibitor, increased HSP70, phosphorylated HSF1
and Hspa1a promoter activity in Caco-2 cells. This suggests that propionate and butyrateinduced upregulation of HSP70 is partly mediated by HDAC inhibition.

6. General conclusion
Fermentable DFs such as GG and PHGG increased the colonic HSP70, possibly through the
SCFA production by intestinal microorganisms. In particular, propionate and butyrate, but not
acetate activate the transcriptional regulation of HSP70 via HSF1 phosphorylation. At least,
two kinases, MEK and mTOR, are responsible for the HSF1 phosphorylation. In addition,
HDAC inhibition also seems to be involved in the HSP70 expression. HSP70 expression
possibly increases the integrity and viability of intestinal epithelial cells, contributing to the
symbiosis with intestinal microorganisms and maintaining intestinal homeostasis. The
increased colonic HSP70 could be involved in the fermentable DFs-mediated benefits to
intestinal health.