Role of dermatopontin in re-epithelialization: implications on keratinocyte migration and proliferation

Abstract

Re-epithelialization is a key event in wound healing and any impairment in that process is associated with various pathological conditions. Epidermal keratinocyte migration and proliferation during re-epithelialization is largely regulated by the cytokines and growth factors from the provisional matrix and dermis. Extracellular matrix consists of numerous growth factors which mediate cell migration via cell membrane receptors. Dermatopontin (DPT), a non-collagenous matrix protein highly expressed in dermis is known for its striking ability to promote cell adhesion. DPT also enhances the biological activity of transforming growth factor beta 1 which plays a central role in the process of wound healing. This study was designed to envisage the role of DPT in keratinocyte migration and proliferation along with its mRNA and protein expression pattern in epidermis. The results showed that DPT promotes keratinocyte migration in a dose dependant fashion but fail to induce proliferation. Further, PCR and immunodetection studies revealed that the mRNA and protein expression of DPT is considerably negligible in the epidermis in contrast to the dermis. To conclude, DPT has a profound role in wound healing specifically during re-epithelialization by promoting keratinocyte migration via paracrine action from the underlying dermis.

Figure 1. Effect of DPT on keratinocytes migration.

Graphical illustration of the percentage of wound area recovered with various concentrations of DPT treatment. ** P-Value (0.0258) was calculated using student's t-test for the indicated concentration by comparing with the untreated cells at the same time point.

Figure 2. Effect of DPT on wounded keratinocyte monolayer.

Representative microscopic images showing the migratory pattern in (a & c) untreated and (b & d) treated cells at 0 (a & b) and 8 (c & d) hours. Scale bar – 100 μm.

Figure 3. Phalloidin staining of cells treated with and without DPT.

The number of thick and extended lamellipodia forming cells was higher in DPT treated cells (a - d) than in control cells (e - h). A typical representation of images are (a & e) Phase contrast image showing the cytoplasmic protrusions formed. (b & f) F-Actin filaments stained with Phalloidin (green) showing the thick actin network formed at the migrating edge, a typical phenotype of the migratory cells. (c & g) Nucleus stained with DAPI (blue). (d & h) Fused image showing the stained nucleus and F-actin. The focal adhesion points formed in the direction of migration are indicated by red arrows in the subset of image b. Scale bar – 50 μm.

Figure 4. Phase contrast images showing lamellipodia formation after scratch assay experiment.

(a) Untreated cells and (b) DPT (500 pg/mL) treated cells. (c) Graphical representation of number of lamellipodia formed in cells with and without DPT treatment. The images were captured after fixing the cells. The lamellipodia observed in cells are indicated with arrows. Scale bar – 100 μm.

Figure 5. Effect of DPT on Keratinocyte growth.

Graphical depiction of the proliferative potential of DPT on keratinocytes assessed through MTT assay. The values represent the mean of three repeated experiments with triplicates for each concentration.

Figure 6. Expression of DPT in epidermis and dermis.

(a) Agarose gel images showing DPT PCR products, 168 bp. (i–v) and RPL32 PCR products, 147 bp. (vii–xi). Lanes: i & xi) No template control for DPT and RPL32 respectively, ii & x) NSF, iii & ix) HaCaT cells, iv & viii) Epidermis, v & vii) Dermis, vi) 100 bp DNA ladder. (b) Western blotting analysis of protein homogenates from i) HaCaT cells and ii) epidermis revealing the absence of DPT protein in contrary to the prominent expression in v) dermal and vi) NSF protein extracts. The bands were compared with iv) rDPT and iii) protein molecular weight marker to determine the band size.

Figure 7. Immunohistochemical analysis of normal skin for expression and localization of DPT protein.

(a, b & c) Sections treated with anti-human DPT primary antibody at different magnifications. (d, e & f) Position-matched control sections without primary antibody treatment at different magnifications. The brown color developed due to HRP-DAB reaction is indicated by arrows. Scale bar – 100 μm.

Figure 8. Immunofluorescent detection of DPT protein in normal skin sections.

(a) Sections treated with FITC conjugated secondary antibody showing strong signals in the dermis (indicated by arrows). ii) Magnified image of the same section revealing the absence of DPT protein in the epidermis. Scale bar – 100 μm.