The Role And Mechanisms Of CD9 In Regulation Of Keratinocytes Migration During Wound Healing

BackgroundWound healing represents a dynamic and well-ordered biological process. Re-epithelialization, an essential step for wound healing, involves the migration of epidermal keratinocytes over the wound site. CD9 has been reported to be expressed in skin epidermis. Our previous in vivo study revealed that CD9 is involved in the regulation of skin wound healing as wound repair is delayed in CD9-null mice. However, plenty of intracellular and intercellular pathways will be activated and coordinated after an injury. Besides, many cell types undergo marked changes in gene expression and phenotype, leading to cell proliferation, migration and differentiation. Therefore, it remains unclear whether CD9 plays a role in wound healing through the regulation of keratinocytes migration and its corresponding signal pathways.Increasing evidences suggest that matrix metalloproteinase-9(MMP-9) also contributes to keratinocyte migration during wound repair. Moreover, plenty of data revealed that metalloproteinases are regulated by CD9.We previously found that CD9 downregulation contributes to keratinocyte migration via MMP-9 activation. The best characterized interactions of tetraspanins are those with integrins, which are involved in signaling pathway. CD9 also shows well-ordered change during wound repair and is implicated in many integrin regulations. Moreover, αvβ6 integrin is revealed to promote corneal wound repair, upregulate MMP-9, and promote cell migration in normal oral keratinocytes. Re-epithelialization is associated with a switch from αvβ5 to αvβ6 during cutaneous wound healing. Besides, during keratinocyte stratification and wound healing, keratinocytesundergo a switch between differentiation and motility. We have previously demonstrated that the expression of CD9 was changed in different wound stages and involved in the regulation of keratinocyte migration.Although CD9 is involved in wound re-epithelialization and CD9 expression in the migrating epidermis is downregulated following wounding and then increases, reachin g levels comparable to that observed in normal skin epidermis when re-epithelialization is complete, little is known about how CD9 expression is regulated during wound healing. Moreover, the wound microenvironment is virtually devoid of oxygen after acute tissue injury and the temporal appearance of hypoxia correlates well with the downregulation of CD9 expression during wound repair.Therefore, this study was undertaken to observe the effect of CD9 in keratinocytes migration and the role of integrins in CD9-regulated MMP-9 activation and keratinocytes migration during wound repair. In addition, the role of CD9 in the switch between keratinocytes differentiation and motility was also detected. Moreover, hypoxia was identified to be involved in the regulation of CD9 expression and keratinocyte migration during wound repair. As such, the research may provide novel insights into mechanisms of tetraspanin CD9 regulation and keratinocyte migration during wound healing.Methods1. Mice were punched and skin histological sections were obtained after punch injury to make a wound repair model in vivo, and confluent HaCa T cells and normal human keratinocyte(NHK) monolayers were scratched to make a in vitrowound repair model. IHC staining was used to detect the change of CD9 expression in migrating keratinocytes at the wound edge during wound repair. Recombinant adenovirus vectors for silencing CD9(CD9-sh RNA) and overexpressing CD9(Ad-CD9) were constructed and used to infect keratinocytes, then the scratch wound assay and the transwellmigration assay were used to observe the impact of CD9 on keratinocyte migration. Besides, the activities of MMP-9 and MMP-2 in supernatants of keratinocyte cultures were assessed by gelatin zymography, while the protein expression of MMP-9 and MMP-2 were analyzed by western blot.Selective MMP-9 inhibitor or JNK inhibitor SP600125(10μM) was also used to test the role of MMP-9 in CD9-regulated keratinocyte migration or the role of JNK signaling in CD9-regulated MMP-9 expression.2. HaCaT cells were infected with recombinant adenovirus vectors to silence CD9 or overexpress CD9 and then the expressions of integrin subunits in CD9-regulated HaCa T cells were analyzed using western blot. The plasma membrane of HaCaT cells was isolated andthe complex was analyzed by immunoprecipitation. The immunofluorescence staining and flow cytometry assay were used to observe the distribution of αvβ5 and αvβ6 at the plasma membrane of keratinocytes. Functional blocking antibodies 10D5 and P1F6(10μg/ml) were used to reveal the role of integrins αvβ5 and αvβ6 in CD9-regulated MMP-9 activation and keratinocyte migration.3. HaCa T cells and primary mouse keratinocytes(MKs) were treated with high Ca2+(1.2mM) for 24 hours to induce differentiation and then the CD9 expression was detected by western blot analysis. The trace of single keratinocyte on FN was monitored by time-lapse phase contrast videomicroscopy and analyzed by ImageJ software. CD9-overexpressed or-silenced HaCaT cells or MKs were cultured in serum-free medium containing 0.03 mM(low concentration) or 1.2mM(high concentration) Ca2+ for 24 hours respectively. The influence of CD9 on cell diameter and spreading area was assessed. The expression profiles of differentiation markers(CK1, CK10, loricrin and filaggrin) in keratinocytes were examined using western blot. Fluorescence immunostaining was used to show the signal of E-cadherin at cell-cell contacts. Western blot analysis and co-immunoprecipitation were used to show the activation of PI3K/Akt signaling in CD9-regualted keratinocytes. CD9-overexpressed MKs cultured in low Ca2+ condition were transfected with si RNA for E-cadherin or pretreated with PI3 K inhibitor LY294002 to determine whether the recruitment of E-cadherin complex to the plasma membrane may account for CD9-mediated keratinocyte differentiation and motility. CD9-silenced MKs treated with JNK inhibitor SP600125 was used to show the role of JNK pathway in the localization of E-cadherin at plasma membrane. Immunostainning also used to detect the distribution of CK10 and E-cadherin at the plasma membrane in CD9-deficient skin.4. HaCaT cells and MKs were subjected to hypoxia condition for 12 hours, 24 hours and 36 hours. The changes ofCD9 levels were analyzed using western blot and real-time PCR. Recombinant adenovirus vectors for overexpressing CD9 and silencing CD9 were constructed and used to infect HaCaT cells prior to hypoxia treatment. The effect of CD9 on hypoxic Ha Ca T keratinocyte migration was evaluated using the scratch wound assay and time-lapsevideomicroscopy. Phosphorylated p38(p-p38) and p38 were tested using western blot. Hypoxic keratinocytes were treated with or without the p38/MAPK inhibitor, SB203580 and the MKK6(Glu) recombinant adenovirus. Then the CD9 expression changes were analyzed using western blot and the hypoxic keratinocyte migration was detected using the cell scratch wound assay and the cell migration assay.Results1.The expression of CD9 was downregulated in migrating keratinocytes during wound repair in vivo and in vitro. Downregulation of CD9 promoted keratinocyte migration in vitro, whereas CD9 overexpression inhibited cell migration. Moreover, CD9 inversely regulated the activity and expression of MMP-9 in keratinocytes, which was involved in CD9-regulated keratinocyte migration. Importantly,CD9silencing-activated JNK signaling was accompanied by the upregulation of MMP-9 activity and expression. Coincidentally, SP600125 decreased the activity and expression of MMP-9 of CD9-silenced Ha CaT cells.2.The expressions of integrin subunits β5 and β6 were regulated by CD9. Furthermore, CD9 silencing triggered the switch from αvβ5 to αvβ6 integrin in Ha Ca T keratinocytes and CD9 overexpression reversed the switch. Importantly, integrin αvβ6 functional blocking antibody 10D5 significantly inhibited CD9 silencing-induced keratinocyte migration and MMP-9 activation.3. CD9 expression was increased in both human and mouse keratinocytes undergoing differentiation. CD9 overexpression in keratinocytes stimulated terminal differentiation, while reduced the cell motility. CD9 silencing inhibited the calcium-induced keratinocyte differentiation and increased the cell motility. Furthemore, CD9 overexpression recruited E-cadherin to the plasma membrane and subsequantly activated PI3K/Akt signaling, while CD9 knockdown inhibited the recruitment of E-cadherin to the plasma membrane and PI3K/Akt activation. Importantly, the silencing of E-cadherin or inhibition of PI3K/Akt signaling reversed the CD9 overexpression-induced differentiation and-reduced motility.4. CD9 expression was downregulated and keratinocyte migration was increased under hypoxic conditions(2% O2). In addition, CD9 overexpression reversed hypoxia-induced cell migration. Hypoxia activated the p38/MAPK pathway. SB203580, a p38/MAPK inhibitor, increased CD9 expression and inhibited keratinocyte migration under hypoxia, while MKK6(Glu) overexpression decreased CD9 expression and promoted hypoxic keratinocyte migration.Conclusion1. CD9 is downregulated in migrating keratinocytes in vivo and in vitro, and a low level of CD9 promotes keratinocyte migration in vitro, in which the regulation of MMP-9 through the JNK pathway plays an important role.2. The switch from αvβ5 to αvβ6 integrin plays a key role in CD9-regulated cell migration and MMP-9 activation in keratinocytes.3. CD9 acts as an important node that regulates the keratinocyte differentiation and motility. The recruitment of E-cadherin to the plasma membrane and activation of PI3K/Akt mediated by CD9 play an important role in these processes.4. Hypoxia regulates CD9 expression and CD9-mediated keratinocyte migration via the p38/MAPK pathway.These findings may potentially introduce a new viewpoint on understanding the underlying mechanism surrounding the biological function of CD9 in keratinocytes during wound healing. In addition, our results may provide potential clinical therapeutic targets for wound repair.