Inhibition of type I procollagen synthesis by damaged collagen in photoaged skin and by collagenase-degraded collagen in vitro

Abstract

Type I and type III procollagen are reduced in photodamaged human skin. This reduction could result from increased degradation by metalloproteinases and/or from reduced procollagen synthesis. In the present study, we investigated type I procollagen production in photodamaged and sun-protected human skin. Skin samples from severely sun-damaged forearm skin and matched sun-protected hip skin from the same individuals were assessed for type I procollagen gene expression by in situ hybridization and for type I procollagen protein by immunostaining. Both mRNA and protein were reduced ( approximately 65 and 57%, respectively) in photodamaged forearm skin compared to sun-protected hip skin. We next investigated whether reduced type I procollagen production was because of inherently reduced capacity of skin fibroblasts in severely photodamaged forearm skin to synthesize procollagen, or whether contextual influences within photodamaged skin act to down-regulate type I procollagen synthesis. For these studies, fibroblasts from photodamaged skin and matched sun-protected skin were established in culture. Equivalent numbers of fibroblasts were isolated from the two skin sites. Fibroblasts from the two sites had similar growth capacities and produced virtually identical amounts of type I procollagen protein. These findings indicate that the lack of type I procollagen synthesis in sun-damaged skin is not because of irreversible damage to fibroblast collagen-synthetic capacity. It follows, therefore, that factors within the severely photodamaged skin may act in some manner to inhibit procollagen production by cells that are inherently capable of doing so. Interactions between fibroblasts and the collagenous extracellular matrix regulate type I procollagen synthesis. In sun-protected skin, collagen fibrils exist as a highly organized matrix. Fibroblasts are found within the matrix, in close apposition with collagen fibers. In photodamaged skin, collagen fibrils are shortened, thinned, and disorganized. The level of partially degraded collagen is approximately 3.6-fold greater in photodamaged skin than in sun-protected skin, and some fibroblasts are surrounded by debris. To model this situation, skin fibroblasts were cultured in vitro on intact collagen or on collagen that had been partially degraded by exposure to collagenolytic enzymes. Collagen that had been partially degraded by exposure to collagenolytic enzymes from either bacteria or human skin underwent contraction in the presence of dermal fibroblasts, whereas intact collagen did not. Fibroblasts cultured on collagen that had been exposed to either source of collagenolytic enzyme demonstrated reduced proliferative capacity (22 and 17% reduction on collagen degraded by bacterial collagenase or human skin collagenase, respectively) and synthesized less type I procollagen (36 and 88% reduction, respectively, on a per cell basis). Taken together, these findings indicate that 1) fibroblasts from photoaged and sun-protected skin are similar in their capacities for growth and type I procollagen production; and 2) the accumulation of partially degraded collagen observed in photodamaged skin may inhibit, by an as yet unidentified mechanism, type I procollagen synthesis.

Figure 1.

Histological and ultrastructural appearance of collagen fibers and dermal fibroblasts in severely photodamaged skin and sun-protected hip skin. Top: Light microscopy of Toluidine blue-stained thick sections. Bottom: Transmission electron microscopy. A and C: Sections of sun-protected hip skin. B and D: Sections of severely sun-damaged forearm skin. Original magnifications: ×160 (A and B), ×4,600 (C and D).

Figure 2.

Degraded collagen is increased in severely photodamaged forearm skin compared to sun-protected hip skin. Values shown represent amounts of hydroxyproline released per mg of homogenized tissue after treatment with α-chymotrypsin ± standard errors (n = 9). Statistical significance was determined using the Student’s t-test. ***, P < 0.001.

Figure 3.

Type I procollagen (α1) mRNA expression is reduced in cells in severely photodamaged forearm skin compared to sun-protected hip skin. Values shown represent average numbers of type I procollagen (α1) mRNA-positive cells per section ± standard errors (n = 7). Statistical significance was determined using the Student’s t-test. *, P < 0.05. Inset: Examples of forearm and hip skin. Original magnification, ×160.

Figure 4.

Type I procollagen (α1) protein expression is reduced in cells in severely photodamaged forearm skin compared to sun-protected hip skin. Values shown represent relative amount of cellular and extracellular staining ± standard errors (n = 9). Statistical significance was determined using the Student’s t-test. *, P < 0.05. Inset: Examples of forearm and hip skin. Original magnification, ×160.

Figure 5.

Proliferation and type I procollagen synthesis by fibroblasts from severely photodamaged forearm skin and sun-protected hip skin are similar. Cell number values (top) are averages ± standard errors, based on 22 and 29 isolates from forearm and hip, respectively. Collagen synthesis values (bottom) represent average amounts of type I procollagen secreted into 1 ml of culture medium (normalized to 8 × 10 cells) during a 1-hour incubation period ± standard errors, based on 12 and 23 isolates from forearm and hip, respectively. Statistical significance was determined by Student’s t-test. Values from the two groups were not different from one another at the P < 0.05 level.

Figure 6.

Degradation of monomeric collagen by bacterial collagenase and human skin collagenase. Intact collagen and collagenase-treated collagen were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. α₁(I) and α₂(I) bands were quantified (together) by densitometry scanning after staining with Coomassie brilliant blue. Values for intact collagen were set at 1.0 and the others valued normalized to this. Inset: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showing the appearance of intact and degraded collagen. Note: the major difference between the two enzyme preparations is the production of characteristic one-quarter-size and three-quarter-size fragments by the human skin collagenase but not the bacterial collagenase.

Figure 7.

Histological and ultrastructural appearance of collagen fibers and dermal fibroblasts on polymerized intact collagen and polymerized collagen after partial degradation by collagenolytic enzymes. Top: Light microscopy of Toluidine blue-stained thick sections. Bottom: Transmission electron microscopy. A and C: Intact collagen. B and D: Partially degraded collagen. Original magnifications: ×260 (A and B), ×8,000 (C and D).

Figure 8.

Contraction of intact and partially degraded collagen by human dermal fibroblasts. Collagen contraction was assessed at day 2 as described in the Materials and Methods. Values shown represent the average diameter of the contracted collagen ± the differences between duplicate samples and averages in a single experiment. A: Dose responses for the two enzyme preparations. B: Inhibitor sensitivity profile. C: Dermal fibroblast dose response.

Figure 9.

Cell growth and type I procollagen synthesis by human dermal fibroblasts are reduced on partially degraded collagen gels compared to intact collagen gels. A: Cell growth. Values represent the mean number of cells present at day 4 ± standard errors (n = 5 foreskin isolates and 4 adult isolates). B: Type I procollagen synthesis. Values represent average ng of type I procollagen per ml ± standard errors (n = 5 foreskin isolates and 4 adult isolates). Before assay, culture media volume from the control and treated groups were adjusted to a common cell number (based on the cell counts presented in A). Statistical significance was determined using the Student’s t-test. *, P < 0.05; **, P < 0.01.