The Mechanisms By Which JNK Inhibits PIAS1 To Delay Wound Healing In Diabetes

Delayed or unhealed skin wounds is a common complication of diabetes.Although the inflammatory microenvironment is known to affect skin wound healing in diabetes,the underlying mechanisms are poorly understood.Previous studies in our laboratory have showed the significant downregulation of E3 ligase Pias1 led to decreased DDX5SUMOylation,which resulted in reduced Ddx5 protein in diabetic skin wounds.The decrease in cutaneous DDX5 reduced REG3A/Reg IIIγexpression,thus delaying wound healing in diabetes.However,the mechanism of decreased PIAS1 in diabetic skin wounds remains unknown.JNK has been known to downregulate the expression of PIAS1 in adipocytes,and our previous observation has showed that JNK2 is highly activated in diabetic skin wounds.Therefore,we hypothesized that the reduction in PIAS1 in diabetic wounds would be related to JNK2.To test this,we examined whether JNK2 would regulate PIAS1 expression in keratinocytes.We stimulated human keratinocytes with JNK inhibitor or JNK activator,and found that JNK inhibitor significantly promoted PIAS1expression while JNK activator inhibited PIAS1 expression.Moreover,we isolated primary murine keratinocytes from WT and Jnk2^-/-mice and found that PIAS1expression increased significantly after JNK2 was knockout.To further confirm that JNK2 is a major factor to inhibit PIAS1 expression in diabetic skin wounds,we analyzed the expression of PIAS1 in skin wounds of WT and Jnk2^-/-diabetic mice.Compared to WT diabetic mice,PIAS1 is increased significantly in skin wounds of Jnk2^-/-diabetic mice.Finally,we constructed PIAS1 luciferase reporter and transfected with c-Jun,one downstream key molecule of JNK,and found that c-Jun significantly inhibited the acitivity of PIAS1 promoter.All these results demonstrate that JNK2inhibits PIAS1 expression in keratinocytes and in diabetes.Since JNK2 can downregulate the expression of PIAS1,next we analyzed if JNK2would affect DDX5 SUMOylation via regulating PIAS1.We first confirmed that PIAS1promoted DDX5 SUMOylation by CO-IP assay.Next we constructed PIAS1 deficient keratinocytes(PIAS1^-/-Ha Cat)by using CRISPR/CAS9 techniques,and stimulated WT and PIAS1^-/-Ha Cat with JNK inhibitor and CHX.We found that the JNK inhibitor increased the half-life of DDX5,while the deletion of PIAS1 reduced the half-life of DDX5 that was increased by JNK inhibitor.Because DDX5 can regulate REG3A pre-m RNA splicing to promote keratinocyte proliferation,we stimulated WT and PIAS1^-/-Ha Cat with JNK inhibitor and detected REG3A variants through DNA-PAGE.We found that JNK inhibitor and PIAS1 promoted REG3A pre-m RNA splicing,respectively,and JNK regulated REG3A pre-m RNA splicing dependent on PIAS1.Furthermore,in vitro keratinocyte scratching assay demonstrated that JNK2 inhibited PIAS1 to reduce REG3A expression,thus delaying keratinocyte proliferation.In conclusion,our findings suggest that JNK2 activation inhibits PIAS1expression,and the decrease in PIAS1 reduces DDX5 SUMOylation,thus decreasing REG3A expression and delaying wound healing.The highly activated JNK2 in diabetic skin suppresses PIAS1 expression to delay wound healing.Our results reveal an unknown function of JNK2 involved in wound healing by regulating PIAS1,and also provide a new insight into the treatment of diabetic skin wounds.