1. Heat Shock-Induced Matrix Metalloproteinase (MMP)-1 and MMP-3 Are Mediated through ERK and JNK Activation and via an Autocrine Interleukin-6 Loop 
Chi-Hyun Park, Min Jung Lee, Jungmi Ahn, Sangmin Kim, Hyeon Ho Kim, Kyu Han Kim, Hee Chul Eun, Jin Ho Chung 
Journal of Investigative Dermatology 
Volume 123, Issue 6, Pages (December 2004)
DOI: /j X x
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
The mRNA and protein of matrix metalloproteinase (MMP)-1 and MMP-3 but not of MMP-2 were induced by heat shock in a temperature-dependent manner in cultured primary human skin fibroblasts. Primary human foreskin fibroblasts were serum starved for 48 h and treated for 30 min in a water bath at 37°C, 43°C, or 45°C. Fresh culture medium was added and the cells were further incubated for the indicated times. The amounts of MMP-1, MMP-2, and MMP-3 mRNA (A–C) were analyzed 24 h post-treatment by semiquantitative RT-PCR. Each level of MMP-1, MMP-2, and MMP-3 mRNA was normalized versus that of the corresponding GAPDH mRNA. Values represent the means±SEM of data from three independent experiments. The amounts of MMP-1, MMP-2, and MMP-3 proteins released into culture media (D) were analyzed 72 h post-treatment by western blotting (MMP-1 and MMP-3) or zymography (MMP-2). Data shown are representative of four independent experiments. The proteins of MMP-1, MMP-2, and MMP-3 shown here are the proform of each protein.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Heat shock leads to the rapid activations of extracellular signal-mediated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). Primary human foreskin fibroblasts were serum starved for 48 h and treated for 30 min in a water bath at 37°C or at 43°C. Fresh culture media without serum were added and the cells were further incubated for the indicated times. Phosphorylated and total forms of ERK, JNK, and p38 MAPK in total cell lysates were analyzed by western blotting. The data shown are a representative of four independent experiments.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
The role of mitogen-activated protein kinases (MAPK) on heat shock-induced matrix metalloproteinase (MMP)-1, MMP-2, and MMP-3. Primary human foreskin fibroblasts were serum starved for 48 h. After being pre-treated for 1 h with dimethyl sulfoxide (DMSO) only (C; control), 10 μM of U0126 (U; MEK inhibitor), 15 μM of SP (SP; JNK inhibitor), or 15 μM of SB (SB; p38 MAPK inhibitor), the cultured fibroblasts were treated for 30 min in a water bath at 37°C or 43°C. Fresh media containing specific MAPK inhibitor were added and the cells were further incubated for the indicated times. The expression levels of MMP-1, MMP-2, and MMP-3 mRNA (A–C) were analyzed 24 h post-treatment by semi-quantitative RT-PCR. Levels of MMP-1, MMP-2, and MMP-3 mRNA were normalized versus the corresponding GAPDH mRNA. Values represent the means±SEM of data from three independent experiments. The amounts of MMP-1, MMP-2, and MMP-3 proteins released into culture media (D) were analyzed 72 h post-treatment by western blotting (MMP-1 and MMP-3) or zymography (MMP-2). Data shown are representative of four independent experiments. The proteins of MMP-1 and MMP-3 shown here are the proform of each protein. The active forms as well as the proform, of MMP-2, are visible in this case. Sometimes we detect not only the proform, but also the active forms, of MMP-2 by zymography. This may be due to the activation of pro-MMP-2 into active forms by MT1-MMP, since we can detect the proform and the active form of MT1-MMP at basal level in human primary skin fibroblasts (data not shown).
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Time-dependent increases in the levels of interleukin (IL)-6 mRNA and the amounts of IL-6 protein released into culture media following heat shock. Primary human foreskin fibroblasts were serum starved for 48 h and treated for 30 min in a water bath at 37°C or 43°C. Fresh culture medium was added and the cells were incubated for the indicated times. The mRNA levels of IL-6 (A) and the amounts of IL-6 protein in culture media (B) were analyzed by semiquantitative RT-PCR and ELISA, respectively. The data shown are representative of three independent experiments. Values are the means±SEM of data from three independent experiments.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Time-dependent increases in the levels of heat shock protein (HSP)72 mRNA and protein following heat shock. Primary human foreskin fibroblasts were serum starved for 48 h and then treated for 30 min in a water bath at 37°C or 43°C. Fresh culture medium was added and the cells were further incubated for the indicated times. The mRNA levels of HSP72 (A) and the protein levels of HSP72 in total cell lysates (B) were analyzed by semiquantitative RT-PCR and western blotting, respectively. The data shown are representative of three independent experiments. Values are the means±SEM of data from three independent experiments.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
The neutralization of interleukin (IL)-6 by anti IL-6 antibody inhibits heat shock-induced matrix metalloproteinase (MMP)-1 and MMP-3 expression. Primary human foreskin fibroblasts were serum starved for 48 h and treated for 30 min in a water bath at 37°C or 43°C. Fresh culture medium was added with control IgG (20 μg per mL) or anti IL-6 antibody (5 μg per mL, 20 μg per mL) and the cells were further incubated for the indicated times. The amounts of MMP-1, MMP-2, and MMP-3 proteins released into culture media were analyzed 72 h post-treatment by western blotting (MMP-1 and MMP-3) or zymography (MMP-2). Data shown are representative of three independent experiments. The proteins of MMP-1 and MMP-3 shown here are the proform of each protein. The active forms as well as the proform of MMP-2 are visible here.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions