The activity of collagenase-1 is required for keratinocyte migration on a type I collagen matrix

Abstract

We have shown in a variety of human wounds that collagenase-1 (MMP-1), a matrix metalloproteinase that cleaves fibrillar type I collagen, is invariably expressed by basal keratinocytes migrating across the dermal matrix. Furthermore, we have demonstrated that MMP-1 expression is induced in primary keratinocytes by contact with native type I collagen and not by basement membrane proteins or by other components of the dermal or provisional (wound) matrix. Based on these observations, we hypothesized that the catalytic activity of MMP-1 is necessary for keratinocyte migration on type I collagen. To test this idea, we assessed keratinocyte motility on type I collagen using colony dispersion and colloidal gold migration assays. In both assays, primary human keratinocytes migrated efficiently on collagen. The specificity of MMP-1 in promoting cell movement was demonstrated in four distinct experiments. One, keratinocyte migration was completely blocked by peptide hydroxymates, which are potent inhibitors of the catalytic activity of MMPs. Two, HaCaTs, a line of human keratinocytes that do not express MMP-1 in response to collagen, did not migrate on a type I collagen matrix but moved efficiently on denatured type I collagen (gelatin). EGF, which induces MMP-I production by HaCaT cells, resulted in the ability of these cells to migrate across a type I collagen matrix. Three, keratinocytes did not migrate on mutant type I collagen lacking the collagenase cleavage site, even though this substrate induced MMP-1 expression. Four, cell migration on collagen was completely blocked by recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1) and by affinity-purified anti-MMP-1 antiserum. In addition, the collagen-mediated induction of collagenase-1 and migration of primary keratinocytes on collagen was blocked by antibodies against the alpha2 integrin subunit but not by antibodies against the alpha1 or alpha3 subunits. We propose that interaction of the alpha2beta1 integrin with dermal collagen mediates induction of collagenase-1 in keratinocytes at the onset of healing and that the activity of collagenase-1 is needed to initiate cell movement. Furthermore, we propose that cleavage of dermal collagen provides keratinocytes with a mechanism to maintain their directionality during reepithelialization.

Figure 1

Collagenase-1 mRNA is expressed by migrating keratinocytes in contact with dermal collagen. (A) A section of uninjured neonatal foreskin grafted onto a SCID mouse was hybridized with a ³⁵S-labeled antisense RNA probe specific for collagenase-1 mRNA. No detectable signal for collagenase-1 mRNA was seen in the epidermis (E) or the dermis (D) of unwounded skin. Mouse skin and the graft junctions are not seen in this field. Under dark-field illumination, pigmented cells are iridescent and appear purplish. (B) In a section of a biopsy taken 2 d after wounding grafted human neonatal skin, autoradiographic signal for collagenase-1 mRNA was seen in basal keratinocytes at the leading edge of reepithelialization (arrows). Signal was confined to keratinocytes migrating into the wound area (W), under the scab (S), and over the dermal wound bed (D). No signal was detected in intact epidermis (E) or in any cell in the dermis. (C) In a section of pyogenic granuloma, which displays many features of an acute wound (SaarialhoKere et al., 1992), collagenase-1 mRNA was seen in basal keratinocytes (arrows) along the dermal–epidermal junction (arrowheads) at the epidermal front. Collagenase-1–positive basal keratinocytes at the migrating front are in direct contact with fibrillar collagen (arrowheads), which appear as thick iridescent fibers under Nomarski optics. (Inset) Under dark-field illumination, autoradiographic signal for collagenase-1 mRNA is seen in basal keratinocytes demarcated by arrows in the larger photomicrograph. Autoradiographic exposure was 21 d for all sections. Bars: (A and B) 100 μm; (C) 25 μm; (Inset) 50 μm.

Figure 2

Collagenase-1 mRNA is expressed by migrating keratinocytes in culture. (A) Keratinocytes were plated on dishes precoated with native type I collagen and 24 h later were processed for in situ hybridization using a ³⁵S-labeled collagenase-1–specific antisense RNA probe. Signal for collagenase-1 mRNA was seen only in migrating keratinocytes (arrows), whereas no autoradiographic signal was detected in hyperproliferative or differentiating keratinocytes, which appear as blurred foci (f) when viewed from above. Autoradiographic exposure was 14 d. (B–D) Primary human keratinocytes were plated on culture slides coated with a mixture of type I collagen and colloidal gold particles and were fixed 20 h later. With Nomarski optics (B) or under dark-field illumination (C), tracks of keratinocyte migration were seen as areas devoid of gold particles (arrows). (B) Often, single cells were seen migrating along large bundles of collagen fibers. (C) Gold was also removed along the border of keratinocyte foci and in association with cells (arrows) that had apparently migrated from the keratinocyte islands. Under phase (C′), internalized gold particles were seen as dense accumulations in keratinocytes. No internalized gold was evident in cells within foci (large arrows). (D and D′) No migration tracks or areas of lysis around cell foci were seen in cultures treated with SC44463. Bars: (A) 50 μm; (B) 12 μm; (C–D′) 25 μm.

Figure 4

HaCaT migration on native type I collagen is MMP dependent. (A) HaCaT keratinocytes were grown on type I collagen– coated dishes and treated with or without 30 ng/ml EGF. Collagenase-1 accumulation in the medium was assessed 48 h later by ELISA and normalized to total cellular protein. (B) HaCaT cells were plated on collagencoated slides, stimulated with EGF, and 24 h later were processed for in situ hybridization with a collagenase-1 ³⁵S-labeled antisense RNA probe. Only HaCaT cells at the periphery of cell clusters expressed collagenase-1 mRNA. Autoradiographic exposure was 14 d. (C–E) HaCaT cells were plated within cloning cylinders on collagen-coated dishes. After 24 h, the cylinders were removed, and the cells were allowed to migrate on collagen alone or in the presence of EGF for 48, 72, or 96 h. Cells were stained, and the area migrated was quantified by scanning densitometry. (E) During the initial 24-h culture period, some HaCaTs were treated with 100 mM HU to inhibit EGFmediated proliferation. The cylinders were removed, and the cells were given fresh medium with or without 30 ng/ ml EGF or EGF plus 25 μM peptide hydroxymate inhibitor SC44463. After 96 h, cultures were washed and stained, and migration was quantified by image analysis. Migration data for HaCaT cells pretreated with (+HU) or without (−HU) HU are shown. The data in D and E are the means ± SD or triplicate wells and are expressed in arbitrary units relative to 0-h controls. (F) HaCaT keratinocytes were plated on culture slides coated with a mixture of colloidal gold particles and type I collagen (Col) or gelatin (Gel). Cells on collagen-coated chambers were treated with 30 ng/ml EGF. To inhibit collagenase-1 activity, cells were treated with (+) or without (−) collagenase-1 affinity-purified antibody or 25 μM SC44463 and were fixed 20 h later. Keratinocyte migration was quantified as described under Materials and Methods, and the data shown are the means ± SEM of duplicate samples from four experiments.

Figure 4

HaCaT migration on native type I collagen is MMP dependent. (A) HaCaT keratinocytes were grown on type I collagen– coated dishes and treated with or without 30 ng/ml EGF. Collagenase-1 accumulation in the medium was assessed 48 h later by ELISA and normalized to total cellular protein. (B) HaCaT cells were plated on collagencoated slides, stimulated with EGF, and 24 h later were processed for in situ hybridization with a collagenase-1 ³⁵S-labeled antisense RNA probe. Only HaCaT cells at the periphery of cell clusters expressed collagenase-1 mRNA. Autoradiographic exposure was 14 d. (C–E) HaCaT cells were plated within cloning cylinders on collagen-coated dishes. After 24 h, the cylinders were removed, and the cells were allowed to migrate on collagen alone or in the presence of EGF for 48, 72, or 96 h. Cells were stained, and the area migrated was quantified by scanning densitometry. (E) During the initial 24-h culture period, some HaCaTs were treated with 100 mM HU to inhibit EGFmediated proliferation. The cylinders were removed, and the cells were given fresh medium with or without 30 ng/ ml EGF or EGF plus 25 μM peptide hydroxymate inhibitor SC44463. After 96 h, cultures were washed and stained, and migration was quantified by image analysis. Migration data for HaCaT cells pretreated with (+HU) or without (−HU) HU are shown. The data in D and E are the means ± SD or triplicate wells and are expressed in arbitrary units relative to 0-h controls. (F) HaCaT keratinocytes were plated on culture slides coated with a mixture of colloidal gold particles and type I collagen (Col) or gelatin (Gel). Cells on collagen-coated chambers were treated with 30 ng/ml EGF. To inhibit collagenase-1 activity, cells were treated with (+) or without (−) collagenase-1 affinity-purified antibody or 25 μM SC44463 and were fixed 20 h later. Keratinocyte migration was quantified as described under Materials and Methods, and the data shown are the means ± SEM of duplicate samples from four experiments.

Figure 3

Collagenase-1–negative HaCaT keratinocytes migrate on gelatin but not on native type I collagen. HaCaT cells were grown within cloning cylinders on dishes precoated with type I collagen (Col) or gelatin (Gel). The cylinders were removed 24 h later, and cells were allowed to migrate for 48 h. The arrows designate the border of the culture when the cloning cylinder was removed. The micrographs shown are representative of three experiments.

Figure 5

Keratinocyte migration on native type I collagen is MMP dependent. Primary human keratinocyte migration was assessed by the colony dispersion assay. Cells were cultured on collagen with or without peptide hydroxymate compounds SC44463 or SC44201. The data shown are the means ± SD of triplicate wells and are expressed in arbitrary units relative to 0-h controls.

Figure 6

Keratinocytes do not migrate on a collagenaseresistant type I collagen. (A) Primary human keratinocyte migration on native type I collagen or on collagenaseresistant type I collagen was assessed by the colony dispersion assay. After 24 h, the cylinders were removed, and the cells were allowed to migrate for 120 h. Some cultures on native type I collagen were exposed to 25 μM SC44463 during the 120-h migration period. The data are the means ± SD of triplicate wells, and migration is expressed in arbitrary units relative to 0-h controls. (B) Keratinocytes were grown to confluence on gelatin, type I collagen, or mutant type I collagen. Collagenase-1 accumulation in conditioned medium at 72 h was determined by ELISA and normalized to total protein. Gelatin was included as a substrate that does not stimulate collagenase-1 expression in keratinocytes.

Figure 7

Keratinocyte migration on type I collagen is collagenase-1-dependent. (A) Colony dispersion. Primary human keratinocytes were grown for 24 h within cloning cylinders on type I collagen. The cloning cylinders were removed, and cells were allowed to migrate for 120 h on collagen in the presence of affinity-purified collagenase-1 antibody (C′ase-1 Ab) at the indicated dilutions or nonimmune IgG or 50 μg/ml recombinant human TIMP-1. The data presented are means ± SD of values from three separate wells per treatment group, and migration is expressed in arbitrary units relative to 0-h controls. (B) Colloidal gold. Primary human keratinocytes were plated on culture slides coated with colloidal gold particles and type I collagen or gelatin and the indicated titers of affinity-purified collagenase-1 antibody or 25 μM SC44463 and were fixed 20 h later. Keratinocyte migration was quantified as described under Materials and Methods, and the data shown are the means ± SEM of duplicate samples from four experiments. The lower panels are dark-field illuminated micrographs of keratinocytes on collagen alone (C), on collagen plus collagenase-1 antibody at 1:2 dilution (D), or on gelatin plus collagenase-1 antibody at 1:2 dilution (E).

Figure 7

Keratinocyte migration on type I collagen is collagenase-1-dependent. (A) Colony dispersion. Primary human keratinocytes were grown for 24 h within cloning cylinders on type I collagen. The cloning cylinders were removed, and cells were allowed to migrate for 120 h on collagen in the presence of affinity-purified collagenase-1 antibody (C′ase-1 Ab) at the indicated dilutions or nonimmune IgG or 50 μg/ml recombinant human TIMP-1. The data presented are means ± SD of values from three separate wells per treatment group, and migration is expressed in arbitrary units relative to 0-h controls. (B) Colloidal gold. Primary human keratinocytes were plated on culture slides coated with colloidal gold particles and type I collagen or gelatin and the indicated titers of affinity-purified collagenase-1 antibody or 25 μM SC44463 and were fixed 20 h later. Keratinocyte migration was quantified as described under Materials and Methods, and the data shown are the means ± SEM of duplicate samples from four experiments. The lower panels are dark-field illuminated micrographs of keratinocytes on collagen alone (C), on collagen plus collagenase-1 antibody at 1:2 dilution (D), or on gelatin plus collagenase-1 antibody at 1:2 dilution (E).

Figure 8

Collagen-mediated induction of collagenase-1 is α2 integrin dependent. Primary human keratinocytes were plated on collagen-coated slides with or without colloidal gold particles, and 2 h later, blocking antibodies (10 μg/ml) against the indicated integrin subunits were added. 20 h after plating, migration was assessed on gold-coated slides, and cells on slides without gold were harvested for RNA isolation. Collagenase-1 mRNA was assessed by reverse transcriptase–PCR, and densitometry was used to quantify relative signal strength. For both assays, migration area and collagenase-1 mRNA levels were normalized to the levels detected in cells on collagen alone.