Korean J Physiol Pharmacol.  2011 Dec;15(6):345-351. 10.4196/kjpp.2011.15.6.345.

4-phenylbutyric Acid Regulates Collagen Synthesis and Secretion Induced by High Concentrations of Glucose in Human Gingival Fibroblasts

• Lee GH
• Oh HW
• Lim HD
• Lee W
• Chae HJ
• Kim HR

• KMID: 2285421
• DOI: http://doi.org/10.4196/kjpp.2011.15.6.345

Abstract

High glucose leads to physio/pathological alterations in diabetes patients. We investigated collagen production in human gingival cells that were cultured in high concentrations of glucose. Collagen synthesis and secretion were increased when the cells were exposed to high concentrations of glucose. We examined endoplasmic reticulum (ER) stress response because glucose metabolism is related to ER functional status. An ER stress response including the expression of glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), inositol requiring enzyme alpha (IRE-1alpha) and phosphoreukaryotic initiation factor alpha (p-eIF-2alpha) was activated in the presence of high glucose. Activating transcription factor 4 (ATF-4), a downstream protein of p-eIF-2alpha as well as a transcription factor for collagen, was also phosphorylated and translocalized into the nucleus. The chemical chaperone 4-PBA inhibited the ER stress response and ATF-4 phosphorylation as well as nuclear translocation. Our results suggest that high concentrations of glucose-induced collagen are linked to ER stress and the associated phosphorylation and nuclear translocation of ATF-4.

Keyword

ER stress; Collagen; Human gingival fibroblasts; Glucose; 4-PBA

MeSH Terms

Activating Transcription Factor 4
Butylamines
Collagen
Endoplasmic Reticulum
Fibroblasts
Glucose
Humans
Inositol
Peptide Initiation Factors
Phenylbutyrates
Phosphorylation
Transcription Factors
Activating Transcription Factor 4
Butylamines
Collagen
Glucose
Inositol
Peptide Initiation Factors
Phenylbutyrates
Transcription Factors

Figure

• 

  Fig. 1 Glucose increases the secretion of soluble collagen. Cells were exposed to the indicated concentrations of glucose for 1, 2, 3 or 4 days. (A) Cells were exposed to the indicated concentrations of glucose for 3 days. Soluble collagen was then analyzed in the media. (B) Cell lysates were used for immunoblotting with anti-pro-collagen 1 or collagen 1 antibody. (C) Immunoblotting using the medium from cells cultured with 40 mM glucose for the indicated periods. Anti-collagen 1 antibody immunoblot (upper) and a separate Coomassie blue-stained gel (lower) are shown. Data represent means±S.E. (n=3). *p<0.05, significantly different from 5 mM glucose-treated cells; NS, non-specific.

• 

  Fig. 2 High concentrations of glucose increased secretion of soluble collagen in a time-dependent manner. Cells were exposed to the indicated concentrations of glucose for 1, 2, 3 or 4 days. Soluble collagen was analyzed in the media (A). Cells were exposed to a concentration of 40 mM glucose for the same period. Cell lysates were then used for immunoblotting with anti-pro-collagen 1 or collagen 1 antibody (B). Immunoblotting using the medium from cells cultured with 40 mM glucose for the indicated periods with anti-collagen I antibody (C) and separate Coomassie blue staining (C, lower). Data represent means±S.E. (n=4). *p<0.05, significantly different from 5 mM glucose-exposed cells during each same period; NS, non-specific.

• 

  Fig. 3 Glucose induces the ER stress response. Cells were exposed to the indicated concentrations of glucose for 3 days. Immunoblotting was performed with anti-GRP78, CHOP, peIF-2α, eIF-2α, IRE-1α or β-actin antibodies (A). Quantification analysis was performed (right). Cells were exposed to 40 mM glucose for the indicated periods. Immunoblotting was performed with anti-GRP78, CHOP, p-eIF-2α, eIF-2α, IRE-1α or β-actin antibodies (B). Quantification was performed (right). After cells were exposed to the indicated concentrations of glucose for 3 days (C, upper) and separately cells were exposed to 40 mM glucose for the indicated periods (C, lower), nuclear fractions were obtained and immunoblotting was performed with anti-ATF-4 or PARP antibodies. Quantification was performed (right). Data represent means±S.E. (n=3). *p<0.05, significantly different from 5 mM glucose-exposed cells during each same period. #p<0.05, significantly different from 40 mM glucosetreated condition during 0 day.

• 

  Fig. 4 The chemical chaperone, 4-PBA inhibits the ER stress response as well as collagen synthesis and secretion. Cells were exposed to the indicated concentrations of glucose with or without 4-PBA for 3 days. Immunoblotting was performed with anti-GRP78, CHOP, p-eIF-2α, eIF-2α, IRE-1α, pro-collagen I, collagen I or β-actin antibodies (A). Quantification was performed (right). Separately, cells were exposed to the indicated concentrations of glucose with or without 4-PBA for 3 days and nuclear fractions were obtained and immunoblotting was performed with either anti-ATF-4 or PARP antibodies (B). Quantification was performed (right). Soluble collagen was analyzed in media from the cells cultured with 5 or 40 mM glucose for 3 days (C). Immunoblotting was performed with the medium from the cells cultured with 40 mM glucose for 3 days, using anti-collagen I antibody (D) and separately stained with Coomassive blue dye (lower). Data represent means±S.E. (n=5). *p<0.05, significantly different from each indicated concentration of glucose-treated cells without 4-PBA. CBB; Coomassive blue staining.

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