Collagen fragments inhibit hyaluronan synthesis in skin fibroblasts in response to ultraviolet B (UVB): new insights into mechanisms of matrix remodeling

Abstract

UVB irradiation causes characteristic features of skin aging including remodeling of the dermal extracellular matrix. A key feature during this process is the up-regulation of matrix metalloproteinases and cleavage of collagen. Hyaluronic acid (HA), a major component of the dermal matrix, decreases after chronic UVB exposure. However, the factors that govern the decline of HA synthesis during the course of actinic aging are largely unknown. The aim of the present study was to explore whether collagen degradation causes inhibition of HA synthesis in human skin fibroblasts. After treatment of fibroblasts with collagen fragments (CF) in vitro, resolution of the actin cytoskeleton and inhibition of HA secretion occurred because of specific down-regulation of hyaluronan synthase 2 (HAS2) expression. The α(v)β(3)-agonist, RGDS, latrunculin A, and an inhibitor of Rho-activated kinase inhibited HAS2 expression. Conversely, blocking antibodies to α(v)β(3) abolished the down-regulation of HAS2 and the cytoskeletal effects. Furthermore, inhibition of cofilin phosphorylation in response to CF was prevented by α(v)β(3)-blocking antibodies. The key role of ERK signaling was shown by reduced nuclear accumulation of phosphoERK and of ELK-1 phosphorylation in response to CF. In addition, the ERK inhibitor PD98059 reduced HAS2 expression. Also, UVB irradiation of fibroblasts caused down-regulation of HAS2, which was sensitive to matrix metalloproteinase inhibitors and to α(v)β(3)-blocking antibodies. In conclusion, these data suggest that CF activate α(v)β(3)-integrins and in turn inhibit Rho kinase (ROCK) signaling and nuclear translocation of phosphoERK, resulting in reduced HAS2 expression. Therefore, a novel mechanism is presented how proteolytic collagen cleavage may inhibit HA synthesis in dermal fibroblasts during extrinsic skin aging.

FIGURE 1.

Inhibition of HA synthesis by CF. Human skin fibroblasts were incubated for 24 h with CF (125 μg/ml) generated by digestion of type I collagen gels. A, change in morphology of fibroblasts in response to CF, 63× magnification. B, pericellular HA was preserved by acid-formalin/ethanol fixation and stained with biotinylated HABP and streptavidin-FITC, 63× magnification; insets, at 200× magnification. Arrows point at strands of pericellular HA. C, HA secretion into the medium. D, molecular weight distribution of [³H]glucosamine-labeled HA as determined by Sephacryl S1000 chromatography. E, pie chart of relative mRNA expression of HAS isoforms as determined by real time RT-PCR. F, regulation of HAS isoforms in response to CF as determined by real time RT-PCR; n = 3–6, mean ± S.E., *, p < 0.05; ***, p < 0.01.

FIGURE 2.

α_vβ₃-Integrin activation mediates HAS regulation by CF. A, preincubation with the α_vβ₃-blocking antibody LM609 (5 μg/ml, 24 h) inhibited regulation of HAS isoforms by CF as determined by real time RT-PCR, B, HAS isoform expression in response to the α_vβ₃-agonist RGDS (10 μg/ml); n = 3–5, mean ± S.E., *, p < 0.05; x, not detectable. C, α_vβ₃ immunostaining of untreated and CF-treated fibroblasts at 24 h. D, actin stress fibers visualized by phalloidin staining; magnification 63×. Non-immune isotype IgG served as control.

FIGURE 3.

Inhibition of ROCK regulates HAS expression. mRNA expression in human fibroblasts was determined by real time RT-PCR. A, mRNA expression after incubation with latrunculin A (3 μmol/liter, 24 h) or the ROCK inhibitor Y27632 (10 μmol/liter, 24 h). B, the ROCK activator lysophosphatidic acid (LPA, 300 nmol/liter) plus or minus CF was added for 24 h; n = 3–5, mean ± S.E., *, p < 0.05.

FIGURE 4.

CF inhibit cofilin phosphorylation. A, phosphorylated cofilin and cofilin were detected by immunoblotting 24 h after the addition of CF plus or minus α_vβ₃-blocking antibody LM609 (5 μg/ml) or the IgG control (C). B, time course of HAS2 mRNA expression as determined by real time RT-PCR. C, time course of cofilin phosphorylation as evidenced by immunoblotting; n = 3, mean ± S.E., *, p < 0.05.

FIGURE 5.

Inhibition of nuclear ERK1/2 activity by CF. A, the ERK inhibitor PD98059 (10 μmol/liter) inhibited HAS2 mRNA expression as determined by real time PCR. B, ERK and phosphoERK in response to CF as determined in total cell lysates after 24 h. C, nuclear translocation of pERK was visualized and quantified by immunostaining and confocal imaging (63× magnification). For quantitative analysis, four images per slide of three independent experiments were analyzed. Using ImageJ, total pERK and nuclear pERK were measured, and the percentage of nuclear pERK was calculated for each image. Panel C indicates control D, the nuclear ERK substrate pELK-1 was detected by immunoblotting of total cell lysates; n = 3–4, mean ± S.E., *, p < 0.05.

FIGURE 6.

UVB-induced collagen cleavage inhibits HAS2 via α_vβ₃. A–D, human fibroblasts were grown in collagen gels to form dermal equivalents. A and B, time course of collagen neoepitope accumulation in response to UVB (10 mJ/cm²) as determined by immunostaining using collagen 2 3/4C_short polyclonal rabbit antibody. Panel C indicates control. C, MMP1 mRNA expression. D, HAS2 mRNA expression. E and F, three-dimensional cultures of fibroblasts in collagen gels were subjected to irradiation with UVB (100 mJ/cm²). After 24 h, HAS2 mRNA was determined in the presence of the MMP inhibitor I (MMP 1 I., 300 nmol/liter) (E) or the α_vβ₃-blocking antibody LM609 (α_vβ₃ AB,5 μg/ml) or isotype control (Iso, 5 μg/ml) (F). G and H, polymeric collagen (120 μg/ml) was added to the medium of monolayer cultures of human skin fibroblasts (C_S). Subsequently, the cultures were subjected to UVB irradiation (UVB_S, 100 mJ/cm²) in the presence (G) or absence (H) of MMP inhibitor I (MMP 1 I._S, 300 nmol/liter). After 3 days, the conditioned medium was added to a new fibroblast culture in the presence or absence of MMP inhibitor I (300 nmol/liter) (G) or α_vβ₃-blocking antibody LM609 (α_vβ₃ AB_S, 5 μg/ml) or isotype control (Iso_S, 5 μg/ml) (H) for 24 h. Subsequently HAS2 mRNA was determined by real time PCR; n = 3, mean ± S.E., *, p < 0.05.

FIGURE 7.

Schematic drawing of the proposed mechanism of HAS2 down-regulation in response to UVB. UVB induces MMP1 expression and degradation of polymeric collagen to CF. The present data suggest that CF activate α_vβ₃-integrins, which inhibit ROCK activity and cofilin phosphorylation. Consequently translocation of pERK in the nucleus and transcriptional activation of HAS2 are suppressed. FAK, focal adhesion kinase.