Mycosporine-like amino acids stimulate hyaluronan secretion by up-regulating hyaluronan synthase 2 via activation of the p38/MSK1/CREB/c-Fos/AP-1 axis

Abstract

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that critically supports the physicochemical and mechanical properties of the skin. Here, we demonstrate that mycosporine-like amino acids (MAAs), which typically function as UV-absorbing compounds, can stimulate HA secretion from normal human fibroblasts. MAA-stimulated HA secretion was associated with significantly increased and decreased levels of mRNAs encoding HA synthase 2 (HAS2) and the HA-binding protein involved in HA depolymerization (designated HYBID), respectively. Using immunoblotting, we found that MAAs at 10 and at 25 μg/ml stimulate the phosphorylation of the mitogen-activated protein kinase (MAPK) p38, extracellular signal-regulated kinase (ERK)/c-Jun, and mitogen- and stress-activated protein kinase 1 (MSK1) (at Thr-581, Ser-360, and Ser-376, respectively) and activation of cAMP-responsive element-binding protein (CREB) and activating transcription factor 2 (ATF2), but not phosphorylation of JUN N-terminal kinase (JNK) or NF-κB (at Ser-276 or Ser-536, respectively), and increased c-Fos protein levels. Moreover, a p38-specific inhibitor, but not inhibitors of MAPK/ERK kinase (MEK), JNK, or NF-κB, significantly abrogated the increased expression of HAS2 mRNA, accompanied by significantly decreased MAA-stimulated HA secretion. These results suggested that the p38-MSK1-CREB-c-Fos-transcription factor AP-1 (AP-1) or the p38-ATF2 signaling cascade is responsible for the MAA-induced stimulation of HAS2 gene expression. Of note, siRNA-mediated ATF2 silencing failed to abrogate MAA-stimulated HAS2 expression, and c-Fos silencing abolished the increased expression of HAS2 mRNA. Our findings suggest that MAAs stimulate HA secretion by up-regulating HAS2 mRNA levels through activation of an intracellular signaling cascade consisting of p38, MSK1, CREB, c-Fos, and AP-1.

Keywords: AP-1; CREB; MSK1; amino acid; c-Fos; c-Jun N-terminal kinase (JNK); cell signaling; fibroblast; hyaluronan; hyaluronan synthase 2; mycosporine-like amino acids; p38 MAPK; protein phosphorylation.

Figure 1.

Chemical structures of porphyra-334 and shinorine.

Figure 2.

A: effects of MAAs on the secretion level of HA by HDFs in culture. MAAs at 0, 1, 10, or 25 μg/ml were added to cultures of HDFs for 72 h as noted, and the conditioned medium was measured for HA levels by ELISA. Data represent means ± S.D., n = 4; **, p < 0.01 versus control (0 μg/ml). B: effects of MAAs on the viability of HDFs in culture. HDFs were cultured for 72 h in the presence of MAAs at the indicated concentrations, and their viability was evaluated using the MTT assay. **, p < 0.01 versus control (0 μg/ml). Data represent means ± S.D. n = 4. C–: effects of MAAs on DNA synthesis (C) and collagen 1 (D), and elastin (E) mRNA levels in HDFs. DNA synthesis was measured 48 h after incubation with MAAs at the indicated concentrations using a cell proliferation ELISA kit. Collagen and elastin mRNA levels were measured by qRT-PCR 3 h after incubation with MAAs at the indicated concentrations. Data represent means ± S.D., n = 4.

Figure 3.

Effects of MAAs on levels of HAS2 mRNA (A/D), HAS3 mRNA (B), HYBID mRNA (C), and protein (E) in HDFs. HDFs were cultured in the presence or absence of MAAs at 25 μg/ml (A–C) or at 0–50 μg/ml (D and E) for the indicated times (A–C) or 3 h (D) and were then subjected to qRT-PCR analysis for HAS2, HAS3, and HYBID mRNA levels or to Western blotting for HYBID protein levels. Representative immunoblots from three independent experiments are shown (E). Data represent means ± S.D. A: n = 3; B: n = 6; C: n = 5; D: n = 4; E: n = 3; *, p < 0.05; **, p < 0.01 versus the nontreated control (0 μg/ml).

Figure 4.

Effects of transfecting a HAS2 siRNA on the MAA-increased secretion of HA in HDFs. A: effects on HAS2 mRNA levels. B: effects on HA secretion levels. After transfection of HAS2 siRNA, HDFs were cultured in the presence or absence of MAAs at 25 μg/ml for 72 h and then were subjected to qRT-PCR analysis for mRNA levels of HAS2. The conditioned medium was measured at 72 h post-treatment for HA levels by ELISA. Data represent means ± S.D. n = 4; *, p < 0.05; **, p < 0.01 versus control (0 μg/ml) or negative siRNA.

Figure 5.

Effects of MAAs on intracellular signaling cascades of p38 (A), ERK (B), JNK (C), ATF2 (D), MSK1(Thr-581/Ser-360/Ser-376) (E–G), c-Jun (H), NF-κB (Ser-276/Ser-536) (I and J), IκB (K), CREB (L), and c-Fos (M) in HDFs. HDFs were incubated with MAAs or with IL-1α at the indicated concentrations and were harvested at 15 and 30 min or 2 h post-treatment and then immunoblotted with antibodies to β-actin and to phosphorylated or nonphosphorylated signaling factors. K: β-actin was used as a loading control because of undetectable levels of nonphosphorylated IκB protein in IL-1α–treated cells. Representative immunoblots from three independent experiments are shown. Data represent means ± S.D., n = 3; *, p < 0.05; **, p < 0.01 versus control (0 μg/ml).

Figure 5.

Effects of MAAs on intracellular signaling cascades of p38 (A), ERK (B), JNK (C), ATF2 (D), MSK1(Thr-581/Ser-360/Ser-376) (E–G), c-Jun (H), NF-κB (Ser-276/Ser-536) (I and J), IκB (K), CREB (L), and c-Fos (M) in HDFs. HDFs were incubated with MAAs or with IL-1α at the indicated concentrations and were harvested at 15 and 30 min or 2 h post-treatment and then immunoblotted with antibodies to β-actin and to phosphorylated or nonphosphorylated signaling factors. K: β-actin was used as a loading control because of undetectable levels of nonphosphorylated IκB protein in IL-1α–treated cells. Representative immunoblots from three independent experiments are shown. Data represent means ± S.D., n = 3; *, p < 0.05; **, p < 0.01 versus control (0 μg/ml).

Figure 5.

Effects of MAAs on intracellular signaling cascades of p38 (A), ERK (B), JNK (C), ATF2 (D), MSK1(Thr-581/Ser-360/Ser-376) (E–G), c-Jun (H), NF-κB (Ser-276/Ser-536) (I and J), IκB (K), CREB (L), and c-Fos (M) in HDFs. HDFs were incubated with MAAs or with IL-1α at the indicated concentrations and were harvested at 15 and 30 min or 2 h post-treatment and then immunoblotted with antibodies to β-actin and to phosphorylated or nonphosphorylated signaling factors. K: β-actin was used as a loading control because of undetectable levels of nonphosphorylated IκB protein in IL-1α–treated cells. Representative immunoblots from three independent experiments are shown. Data represent means ± S.D., n = 3; *, p < 0.05; **, p < 0.01 versus control (0 μg/ml).

Figure 5.

Effects of MAAs on intracellular signaling cascades of p38 (A), ERK (B), JNK (C), ATF2 (D), MSK1(Thr-581/Ser-360/Ser-376) (E–G), c-Jun (H), NF-κB (Ser-276/Ser-536) (I and J), IκB (K), CREB (L), and c-Fos (M) in HDFs. HDFs were incubated with MAAs or with IL-1α at the indicated concentrations and were harvested at 15 and 30 min or 2 h post-treatment and then immunoblotted with antibodies to β-actin and to phosphorylated or nonphosphorylated signaling factors. K: β-actin was used as a loading control because of undetectable levels of nonphosphorylated IκB protein in IL-1α–treated cells. Representative immunoblots from three independent experiments are shown. Data represent means ± S.D., n = 3; *, p < 0.05; **, p < 0.01 versus control (0 μg/ml).

Figure 5.

Effects of MAAs on intracellular signaling cascades of p38 (A), ERK (B), JNK (C), ATF2 (D), MSK1(Thr-581/Ser-360/Ser-376) (E–G), c-Jun (H), NF-κB (Ser-276/Ser-536) (I and J), IκB (K), CREB (L), and c-Fos (M) in HDFs. HDFs were incubated with MAAs or with IL-1α at the indicated concentrations and were harvested at 15 and 30 min or 2 h post-treatment and then immunoblotted with antibodies to β-actin and to phosphorylated or nonphosphorylated signaling factors. K: β-actin was used as a loading control because of undetectable levels of nonphosphorylated IκB protein in IL-1α–treated cells. Representative immunoblots from three independent experiments are shown. Data represent means ± S.D., n = 3; *, p < 0.05; **, p < 0.01 versus control (0 μg/ml).

Figure 6.

Effects of p38, JNK, NF-κB, and MEK inhibitors on the MAA-increased gene expressions of HAS2 and MAA-stimulated secretion of HA in HDFs. HDFs were incubated with MAAs at 25 μg/ml in the presence or absence of signaling inhibitors for 3, 6, or 72 h, and cell lysates or the conditioned medium was subjected to RT-PCR analysis or ELISA, respectively. A: p38 inhibitor/qRT-PCR, 6 h, data represent means ± S.D., n =; *, p < 0.05; **, p < 0.01, versus MAAs (0 or 25 μg/ml). B: p38 inhibitor/ELISA, 72 h, data represent means ± S.D., n = 4; **, p < 0.01 versus MAAs (0 or 25 μg/ml). C: NF-κB and JNK inhibitors/qRT-PCR, 6 h, data represent means ± S.D., n = 5; *, p < 0.05; **, p < 0.01. D: MEK inhibitor/qRT-PCR, 3 h, data represent means ± S.D., n = 4; *, p < 0.05; **, p < 0.01 versus MAAs 0 μg/ml or MEK inhibitor 0 μm.

Figure 7.

Effects of transfecting an ATF2 siRNA or a c-Fos siRNA. A: effect of transfecting an ATF2 siRNA on the expression of ATF2 protein and its phosphorylation. HDFs were incubated with or without MAAs at 25 μg/ml. Lysates were harvested at 15 min post-treatment and were immunoblotted with antibodies to β-actin and to phosphorylated or nonphosphorylated ATF2. Representative immunoblots from three independent experiments are shown. Data represent means ± S.D., n = 3; **, p < 0.01. B: effect of transfection of a c-Fos siRNA on the MAA-stimulated expressions of c-Fos protein. HDFs were incubated with or without MAAs at 25 μg/ml. Lysates were harvested at 2 h post-treatment and were immunoblotted with antibodies to β-actin and c-Fos. Representative immunoblots from three independent experiments are shown. Data represent means ± S.D., n = 3; *, p < 0.05; **, p < 0.01. C: effects of transfection of an ATF2 siRNA or a c-Fos siRNA on MAA-increased gene expression of HAS2 in HDFs. After transfection of an ATF2 siRNA or a c-Fos siRNA, HDFs were cultured in the presence or absence of MAAs at 25 μg/ml for 3 h and then were subjected to RT-PCR analysis for mRNA levels of HAS2. Data represent means ± S.D., n = 3; *, p < 0.05; **, p < 0.01.

Figure 8.

MAA-activated signaling cascades leading to the increased secretion of HA.