Korean J Physiol Pharmacol.  2015 May;19(3):241-247. 10.4196/kjpp.2015.19.3.241.

p-Coumaric Acid Attenuates UVB-Induced Release of Stratifin from Keratinocytes and Indirectly Regulates Matrix Metalloproteinase 1 Release from Fibroblasts

• Seok JK
• Boo YC

• KMID: 1791425
• DOI: http://doi.org/10.4196/kjpp.2015.19.3.241

Abstract

Ultraviolet (UV) radiation-induced loss of dermal extracellular matrix is associated with skin photoaging. Recent studies demonstrated that keratinocyte-releasable stratifin (SFN) plays a critical role in skin collagen metabolism by inducing matrix metalloproteinase 1 (MMP1) expression in target fibroblasts. In the present study, we examined whether SFN released from UVB-irradiated epidermal keratinocytes increases MMP1 release from dermal fibroblasts, and whether these events are affected by p-coumaric acid (p-CA), a natural phenolic compound with UVB-shielding and antioxidant properties. HaCaT cells were exposed to UVB in the absence and presence of p-CA, and the conditioned medium was used to stimulate fibroblasts in medium transfer experiments. The cells and media were analyzed to determine the expressions/releases of SFN and MMP1. UVB exposure increased SFN release from keratinocytes into the medium. The conditioned medium of UVB-irradiated keratinocytes increased MMP1 release from fibroblasts. The depletion of SFN using a siRNA rendered the conditioned medium of UVB-irradiated keratinocytes ineffective at stimulating fibroblasts to release MMP1. p-CA mitigated UVB-induced SFN expression in keratinocytes, and attenuated the MMP1 release by fibroblasts in medium transfer experiments. In conclusion, the present study demonstrated that the use of UV absorbers such as p-CA would reduce UV-induced SFN-centered signaling events involved in skin photoaging.

Keyword

Matrix metalloproteinase-1; Skin photoaging; Stratifin; UV

MeSH Terms

Collagen
Culture Media, Conditioned
Extracellular Matrix
Fibroblasts*
Keratinocytes*
Matrix Metalloproteinase 1*
Metabolism
Phenol
RNA, Small Interfering
Skin
Collagen
Culture Media, Conditioned
Matrix Metalloproteinase 1
Phenol
RNA, Small Interfering

Figure

• 

  Fig. 1 Effects of UVB irradiation SFN expression/release of HaCaT cells. HaCaT cells were irradiated with UVB at 5~15 mJ cm-2 and incubated for 24 h. (A) SFN mRNA levels were determined by qRT-PCR analysis using GAPDH as a control. (B) SFN protein levels were determined by Western blot analysis of whole cell lysates using β-actin as a control. (C) The released SFN protein levels were determined by Western blot analysis of the conditioned media derived from HaCaT cells exposed to different doses of UVB. For detection of total protein, the transferred membrane was stained with Ponceau S. Data are presented as fold changes versus the non-irradiated control (Mean±SE, n=3). *p<0.05 versus non-irradiated controls.

• 

  Fig. 2 Effects of the conditioned medium derived from UVB-irradiated HaCaT cells on fibroblast expression/release of MMP1 and PIP. Human epidermal fibroblasts were seeded and cultured in growth medium for 24 h then the medium was replaced with the conditioned medium derived from HaCaT cells exposed to UVB at 5~15 mJ cm-2. After a 24 h incubation period, MMP1 expression was determined at the mRNA by qRT-PCR (A), and at the protein levels by western blots (B). The levels of MMP1 (C) and PIP (D) in the conditioned medium of fibroblasts were determined using sandwich immunoassay kits. Data are presented as fold changes versus the control cells without medium transfer (mean±SE, n=3). *p<0.05 versus controls.

• 

  Fig. 3 An essential role for SFN derived from UVB-irradiated HaCaT keratinocytes in stimulating fibroblasts to express/release MMP1 protein. (A, B) HaCaT cells were transfected with a SFN siRNA or negative control siRNA for 24 h, followed by exposure to UVB at 15 mJ cm-2. After a 24 h incubation period, the intracellular (A) and released SFN protein levels (B) were determined by western blots of whole cell lysates and the conditioned media, respectively. For detection of total protein, transferred membrane was stained with Ponceau S. (C, D) Fibroblasts were cultured for 24 h in their own growth medium or the conditioned medium derived from HaCaT cells. The medium was then replaced by the fibroblasts growth medium without serum. After 24 h of incubation, MMP1 (C) and PIP (D) in the conditioned medium of fibroblasts were quantified using sandwich immunoassay kits. Data are presented as fold changes versus the control cells without medium transfer (Mean±SE, n=3). *p<0.05; n.s., not significant.

• 

  Fig. 4 Effects of p-CA on the UVB-induced cytotoxicity and caspase 3 activation in HaCaT cells. HaCaT cells were irradiated with UVB at 15 mJ cm-2 or not, in the absence or the presence of p-CA at the indicated concentrations and incubated for 24 h. (A) Cell viability was determined using an MTT assay. (B) Whole cell lysates of equal amounts of proteins were used for the western blot analysis of the active cleaved and inactive proforms of caspase 3. Data are presented as % of control or fold changes versus the control (Mean±SE, n=3). *p<0.05.

• 

  Fig. 5 Effects of p-CA on the UVB-induced SFN expression/release of HaCaT cells. HaCaT cells were irradiated with UVB at 15 mJ cm-2 or not, in the absence or the presence of p-CA at the indicated concentrations. The cells were then cultured in the fresh medium without p-CA for 24 h to harvest the cells and the conditioned medium. (A) SFN mRNA levels were determined by qRT-PCR analysis using GAPDH as a control. (B) The released SFN protein levels were determined by western blot analysis of the conditioned media derived from HaCaT cells. For detection of total protein, the transferred membrane was stained with Ponceau S. Data are presented as fold changes versus the control cells (mean±SE, n=3). *p<0.05.

• 

  Fig. 6 Effects of the conditioned medium of HaCaT cells UVB-irradiated in the presence of p-CA on the releases of MMP1 and PIP from fibroblasts. HaCaT cells were irradiated with UVB at 15 mJ cm-2 or not, in the absence or the presence of p-CA at the indicated concentrations. The cells were then cultured in the fresh medium without p-CA for 24 h to harvest the conditioned medium. Fibroblasts were cultured for 24 h in the conditioned medium derived from HaCaT cells, and then the medium was replaced with fibroblast growth medium without serum. After incubating for 24 h, MMP1 (A) and PIP (B) in the fibroblasts conditioned medium were quantified using sandwich immunoassay kits. Data are presented as fold changes versus the control cells (mean±SE, n=3). *p<0.05.

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