Ann Dermatol.  2010 Nov;22(4):379-388. 10.5021/ad.2010.22.4.379.

Cholesterol, a Major Component of Caveolae, Down-regulates Matrix Metalloproteinase-1 Expression through ERK/JNK Pathway in Cultured Human Dermal Fibroblasts

• Kim S
• Han J
• Lee DH
• Cho KH
• Kim KH
• Chung JH

• KMID: 2266173
• DOI: http://doi.org/10.5021/ad.2010.22.4.379

Abstract

BACKGROUND
Cholesterol is a major component of specialized membrane microdomains known as lipid rafts or caveolae, which modulate the fluidity of biological membranes. Membrane cholesterol therefore plays an important role in cell signaling and vesicular transport.
OBJECTIVE
In this study, we investigated the effects of cholesterol on matrix metalloproteinase-1 (MMP-1) expression in human dermal fibroblasts.
METHODS
MMP-1 mRNA and protein expression were determined by RT-PCR and Western blotting, respectively. AP-1 DNA binding activity was detected by electrophoretic mobility shift assays. The amount of cholesterol was analyzed by cholesterol assay kit.
RESULTS
We observed that MMP-1 mRNA and protein expression was dose-dependently decreased by cholesterol treatment. In contrast, cholesterol depletion by a cholesterol depletion agent, methyl-beta-cyclodextrin (M beta CD) in human dermal fibroblasts, increased MMP-1 mRNA and protein expression in a dose-dependent manner. Also, we investigated the regulatory mechanism of M beta CD-induced MMP-1 expression: cholesterol depletion by M beta CD, activated ERK1/2 and JNK, but not p38 MAPK cascade, and it also significantly increased c-Jun phosphorylation, c-Fos expression and activator protein-1 binding activity. Furthermore, the inhibition of ERK or JNK with specific chemical inhibitors prevented M beta CD-induced MMP-1 expression, which indicates that ERK and JNK play an important role in cholesterol depletion-mediated MMP-1 induction. In addition, M beta CD-induced phosphorylation of ERK and JNK and MMP-1 expression were suppressed by cholesterol repletion.
CONCLUSION
Our results suggest that cholesterol regulates MMP-1 expression through the control of ERK and JNK activity in human dermal fibroblasts.

Keyword

AP-1; Cholesterol; Methyl-beta-cyclodextrin; MMP-1

MeSH Terms

beta-Cyclodextrins
Blotting, Western
Caveolae
Cholesterol
DNA
Electrophoretic Mobility Shift Assay
Fibroblasts
Humans
Matrix Metalloproteinase 1
Membrane Microdomains
Membranes
p38 Mitogen-Activated Protein Kinases
Phosphorylation
RNA, Messenger
Transcription Factor AP-1
Cholesterol
DNA
Matrix Metalloproteinase 1
RNA, Messenger
Transcription Factor AP-1
beta-Cyclodextrins
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Fig. 1 Cholesterol treatment decreased the expression of matrix metalloproteinase-1 (MMP-1) mRNA and protein in cultured human dermal fibroblasts. After serum-starvation for 24 h, cells were treated with cholesterol at the indicated concentrations for 1 h and then further incubated for 24 h (mRNA) and 72 h (protein), respectively. MMP-1 mRNA (cell lysates) and protein (culture media) expression were determined by (A) RT-PCR and (B) Western blotting, respectively. The results shown are representative of three independent experiments. Values shown are means±SEM. *p<0.05, †p<0.01 vs. Control. Con: control, Chol: cholesterol.

• 

  Fig. 2 Depletion of cholesterol increased matrix metalloproteinase-1 (MMP-1) mRNA and protein expression in cultured human dermal fibroblasts. (A, B) After serum-starvation for 24 h, cells were treated with methyl-beta-cyclodextrin (MβCD) at the indicated concentrations for 1 h. Fresh serum-free media were added and cells were further incubated for 24 h (mRNA) and 72 h (protein), respectively. (C) After serum-starvation for 24 h, cells were treated with 10 mM MβCD and further incubated at 37℃ for the indicated times. ERK1/2, JNK, and p38 phosphorylation were measured in whole cell lysates by Western blotting. (D) After serum-starvation for 24 h, cells were treated with 10 mM MβCD for the indicated times at 37℃. AP-1 DNA binding activity was detected by electrophoretic mobility shift assays in nuclear extracts (5µg protein) as described in Materials and Methods. (E) After serum-starvation for 24 h, cells were pretreated with various inhibitors [U0126: U (10µM), SP600125: SP (10µM), SB203580: SB (10µM)] for 30 min prior to MβCD treatment and then incubated without or with 10 mM MβCD for 1 h and further incubated at 37℃ for 72 h. The MMP-1 protein levels in culture media were detected by Western blotting. β-Actin was used as the standard control. The results shown are representative of three independent experiments. Values shown are means±SEM. *p<0.05, †p<0.01 vs. Control. ‡p<0.05 vs. the MβCD-treated cells. Con: control.

• 

  Fig. 3 Cholesterol repletion decreased cholesterol depletion-induced matrix metalloproteinase-1 (MMP-1) mRNA and protein in cultured human dermal fibroblasts. (A, B) After serum-starvation for 24 h, cells were treated for 1 h without or with 10 mM methyl-beta-cyclodextrin (MβCD) in the absence or the presence of 100µg/ml cholesterol. Following addition of fresh media, cells were further incubated at 37℃ for 24 h (mRNA) and 72 h (protein), respectively. MMP-1 mRNA (cell lysates) and protein (culture media) expression were determined by (A) RT-PCR and (B) Western blotting, respectively. (C) After serum-starvation for 24 h, cells were treated with 10 mM MβCD and/or 100µg/ml cholesterol and then further incubated at 37℃ for 30 min. ERK1/2, JNK, and p38 phosphorylation were measured in whole cell lysates by Western blotting. (D) After serum-starvation for 24 h, cells were treated with 10 mM MβCD and/or 100µg/ml cholesterol for the indicated times at 37℃. AP-1 DNA binding activity was detected by electrophoretic mobility shift assays in nuclear extracts (5µg protein) as described in Materials and Methods. The results shown are representative of three independent experiments. Values shown are means±SEM. *p<0.05, †p<0.01 vs. Control, ‡p<0.05 vs. the MβCD-treated cells. Con: control, Chol: cholesterol.

• 

  Fig. 4 The level of cellular cholesterol after methyl-beta-cyclodextrin (MβCD) and/or cholesterol treatment in human dermal fibroblasts. After serum-starvation for 24 h, cells were treated for 1 h without or with 10 mM MβCD. Filipin staining was performed as described in Materials and Methods (A) Control, (B) MβCD, (C) MβCD+Chol. (D) After serum-starvation for 24 h, cells were treated for 1 h without or with 10 mM MβCD. For repletion of cholesterol, cells were treated for 1 h with 100µg/ml cholesterol. The organic extract of cell was solubilized in 1 ml of the cholesterol assay kit buffer solution. Samples were incubated for 1 h at 37℃ prior to measuring absorbance at 505 nm. The results shown are representative of three independent experiments. Values shown are means±SEM. *p<0.05 vs. Control, †p<0.05 vs. the MβCD-treated cells. Con: control, Chol: cholesterol.

• 

  Fig. 5 Schematic model.

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