We show here that insulin, when topically applied to skin excision wounds, accelerates re-epithelialization and stimulates "maturation" of the healing tissue. These effects are dependent on the insulin receptor but independent of EGF/EGF-R; PI3K-Akt-Rac1 signaling pathways are critically involved, and healing is α3 and LN332-dependent.

Histological observation of wounds treated with insulin suggests that this protein stimulates keratinocyte migration. Although the effects of insulin on keratinocyte proliferation are well established [20], its effect on migration of these cells is not clear. To study the latter process, we used HaCaT keratinocytes in culture. Cells were plated in cloning rings and allowed to attach; the rings were removed after marking their positions and the cells were then treated with insulin. Migration distances from the initial edge of the cells to the new edge of the cells were measured at 24, 48, and 72 hr. At each time point, keratinocytes treated with insulin showed increased migration over the control (Fig. 2A, B). To determine whether this effect is dose-dependent, we performed the migration assays with different doses of insulin. A concentration as low as 10^-8 M insulin was able to increase keratinocyte migration, which was highly significant after 48 hr of treatment. However, concentrations ranging from 10^-7 M to 10^-5 M significantly enhanced keratinocyte migration by 24 h (Fig. (Fig.2C).2C). To eliminate the possibility that this migration is dependent on proliferation, we treated the cells with mitomycin C, a potent DNA crosslinker and hence inhibitor of cell proliferation, in the presence or absence of insulin. Cells were pre-treated with 5 μg/ml of mitomycin C for 3 hrs and then exposed to 10^-7 M insulin for 24 and 48 hrs. Although we observed that proliferation was halted, insulin-induced migration was not (Fig. (Fig.2D),2D), strongly suggesting that the two processes are independently regulated by this hormone.

Small GTPases of the RhoA family play important roles in cell motility. Therefore, we tested the possibility that the PI3K-Akt pathway stimulates RhoA activation during insulin-induced keratinocyte migration. Using immunolabeling, we show that there was no significant difference in RhoA distribution shortly after insulin treatment (Fig. 5A–C) nor did a change occur at least for 4 h. However, 3 min after insulin treatment, Rac1, another member of the RhoA family of GTPases, translocated from the cytosol to the plasma membrane, indicating its activation (Fig. (Fig.5E).5E). This redistribution effect was also seen after 5 min of insulin treatment (Fig. (Fig.5F).5F). In addition, plasma membrane ruffling was observed at the leading edge of migrating keratinocytes, with Rac-1 being present in the membrane of ruffles (Fig. (Fig.5F;5F; arrow). To determine whether this translocation is consistent with the activation of Rac1, we performed Rac1 pull down assays, which can specifically pull down the active form of Rac, and found elevated levels of active Rac1 after insulin treatment (Fig. (Fig.5G).5G). In order to confirm that Rac1 activation is important in insulin-induced keratinocyte migration, we transfected keratinocytes with plasmids containing mutant forms of Rac1, and then observed the effects of these mutants on insulin-stimulated cell migration. Keratinocytes were transfected with either the constitutively active form of Rac1 (V12, Rac1-CA), dominant-negative mutant Rac1 (N17, Rac1-DN), or wild type Rac1 (Rac1-WT). 24 h after transfection, scratch wounds were made in the cell cultures, and cell migration distances were measured in non-transfected and transfected cells. After treatment for 24 h, insulin stimulated migration in non-transfected cells, as well as in cells transfected with Rac1-CA or Rac1-WT. Cells transfected with Rac1-CA showed increased migration, even without insulin treatment. However, insulin-induced migration was eliminated in cells transfected with Rac1-DN (Fig. (Fig.5H).5H). To determine whether the PI3K-Akt pathway is required for insulin-induced Rac1 activation/translocation, LY294002 was used to pre-treat the cells before insulin treatment. Insulin-induced Rac1 activation (Fig. (Fig.5I)5I) and translocation (Fig. (Fig.5J)5J) was inhibited by this PI3K inhibitor.

C57BL/6J mice were purchased from The Jackson Laboratory (USA), and housed at the University of California, Riverside (UCR) vivarium. All experimental protocols were approved by the UCR Institutional Animal Care and Use Committee. Experiments were performed using 8–12 wk old mice. Mice were anesthetized with a single intraperitoneal injection of ketamine (80 mg/kg body weight)/xylazine (16 mg/kg body weight). Full-thickness 7 mm punch wounds (excision of the skin and the underlying panniculus carnosus) were made on the back of the mice. The wounds were then treated as indicated for the various experiments and covered with a transparent dressing (Bioclusive, Johnson & Johnson Medical Limited, USA) until day 3 after wounding. After this time, the dressings were removed, and the wounds were exposed. Healing was monitored by taking photographs at the indicated time points. In addition, the wound area was drawn on a transparent plastic film for further comparisons of wound area at each time point. For graphing purposes, wound closure was expressed as percentage of wound area (area of wound measured at any one day post wounding divided by the initial wound area).

At day 3 after wounding, as well as the day that complete healing occurred, animals were anesthetized, and skin samples consisting of the wounded area plus 5 mm of the surrounding unwounded and/or healed skin area were collected. Tissues were fixed in 4% pareformaldehyde for 2 h and incubated in 0.1 M glycine/PBS for 1 h followed by 15% and 30% sucrose before embedding in OCT (Tissue-Tek, Sakura Finetek. USA, Inc), freezing in a slush of ethanol/dry ice, and stored in -80 C. After sample collection, the mice were euthanized using CO₂. The extent of migration of the keratinocyte was measured in sections stained with Hematoxylin and Eosin. The length of migration tongue was determined by the distance from the margin of the wound to the tip of the migrating keratinocytes.

The human keratinocyte cell line HaCaT was cultured in 5% CO₂ at 37°C in DMEM supplemented with 10% FBS, 10 units/ml penicillin, and 10 μg/ml streptomycin sulfate (GIBCO, Invitrogen Corporation).

Cells were treated as indicated, and then washed with ice-cold 1 × PBS, and lysed on ice with lysis buffer containing 0.5% Triton X100, 0.5% Nonidet P-40, 10 mM Tris, pH 7.5, 2.5 mM KCl, 150 mM NaCl, 30 mM b-glycerophosphate, 50 mM NaF, 1 mM Na3VO4, 0.1% SDS and additional protease and phosphatase inhibitor cocktails (Sigma). Protein concentrations were measured using the DC protein assay kit (Bio-Rad). Equal amounts of protein in the cell extracts were mixed with sample buffer, boiled, and analyzed using 10% acrylamide SDS-PAGE. Immunoblotting was performed with the indicated primary Abs and the appropriate HRP-conjugated secondary Abs, followed by incubation with West Dura extended duration substrate (Pierce Biotechnology). Blots were then re-probed for house keeping proteins to show equal loading. For LN332 and integrin α3, protein extracts were prepared as indicated above, but were mixed with non-reducing sample buffer, and, for LN332 detection, samples were boiled. The extracts were then analyzed using 7.5% acrylamide SDS-PAGE, followed by immunoblotting as described above.

We used two types of assays: (i) The cloning ring assay in which 2.0 × 10⁴ HaCaT cells were plated in a cloning ring 6 mm diameter set within a 35-mm cell culture dish. Four hours after seeding, the cylinder was removed, the cell edges were marked, and migration was measured at the indicated times by measuring the distance from the initial cell edge to the edge of the migrating cells; (ii) For the scratch assay the HaCaT cells were plated in cell culture dishes and after reaching confluence, scratch wounds were made as previously described [42]. Briefly, we used the small end of a 1 ml pipet tip to scratch the cells on a 35 mm plate, and then marked the edges of the scratches. Cell migration was measured at the indicated times by measuring the distance from the initial cell edge to the edge of the migrating cells. The scratch assay was used when we performed transfection experiments to avoid trypsinization and re-plating into the cloning ring.

Recombinant adenovirus containing the constitutively active mutant of Akt (Akt-CA), the dominant-negative mutant of Akt (Akt-DN), and the parental adenoviral vector (referred to as null) were gifts from John Shyy (UCR, Biomedical Sciences Division). Cells were infected with various recombinant adenoviruses at 50 moi; media was changed after 5 h of incubation with adenovirus. After 48 h of infection, cells were subjected to further treatment, as indicated in the Results section. Plasmids expressing the constitutively active form of Rac1 (V12, Rac1-CA), the dominant-negative mutant Rac1 (N17, Rac1-DN) and the wild type Rac1 (Rac1-WT; gifts from Dr. Miguel Del Pozo, Centro National de Investigaciones Cardiovasculares, Madrid, Spain) were transfected using Lipofectin (Invitrogen, USA) according to the manufacturer's protocol. Briefly, one day before transfection, the cells were plated in growth media without antibiotics. Transfections were performed at 40–60% cell confluence. 2 μg of DNA in 100 μl of DMEM without serum, were mixed gently with 5 μl Lipofectin in 100 μl of MDEM without serum. This mixture was incubated for 15 min at room temperature and then added to the cells which were then incubated at 37°C and 5% CO₂ for 16 h. At this time the medium was replaced with medium containing serum and analyzed at the appropriate times.

Cells were cultured in chamber slides (Nunc), and fixed in 4% paraformaldehyde for 20 min, rinsed with PBS, incubated in PBS containing 0.1 M glycine for 20 min, and blocked with 3% BSA, 0.1%Triton X-100 in PBS for 30 min. Primary Abs in 1% BSA/PBS were applied to the sample for 2 h at room temperature, washed, and incubated with 1:50 dilution of FITC or Texas red-conjugated secondary Abs for 1 h at room temperature. After washing, the cells were mounted in Vectashield containing DAPI (Vector Laboratories, Inc. Berlingame, CA). Immunofluorescence was visualized and imaged using a Leica SP2 laser scanning confocal microscope. For frozen tissues, 8-μm cryosections were washed in 1 × PBS to remove the OCT, fixed in 2% paraformaldehyde for 10 min, incubated in 0.1 M glycine in 1 × PBS, followed by the primary and secondary Abs using the same procedure as indicated above.