The Role Of Neuregulin-1/ErbB4 Signaling In Cutaneous Wound Repair

Background&ObjectivesSkin is the largest sensory organ within the human body and has a lot of functions in acting as barriers,sensations,immune thermoregulation,and endocrine excretion,and so on.Large-scale skin injury is very common,caused by various acute injuries including burns,skin avulsions,and surgical operations.The incidence of refractory skin wounds,particularly injury-induced pressure ulcers,venous ulcers,and diabetic ulcers,are increased year by year that seriously affect the daily life of patients and bring great challenge to wound repair surgeons.Skin wound repair is a complex and orderly process involving various types of cells,extracellular matrix,cytokines and growth factors.However,the specific cellular mechanism and novel regulatory factors in the process of wound healing remain still obscure.Thus,we here explore a novel regulatory mechanism underlying the skin wound repair from a new perspective.Neuroregulin-1（NRG1）is an extracellular growth factor widely distributed in the central and peripheral nervous system.It has an epidermal growth factor（EGF）-like domain and mainly functions in vivo through activating Erb B tyrosine kinase receptors.The NRG1/Erb Bs signaling have multiple biological functions and play important regulatory roles in cell proliferation,differentiation and migration,as well as neural development the repair and regeneration of heart injury and so on.Among this receptor family,including Erb B2,Erb B3 and Erb B4,the latter is a main target of NRG1.Erb B4 is the only receptor that can directly bind to NRG1 and exert tyrosine kinase activity meanwhile.However,previous research about them is only mainly limited in the nervous or cardiovascular systems.Thus,the expression distribution and role of Erb B4 in the skin are not clear so far.Here,this study will be focused on the role and its specific cellular mechanisms of NRG1/Erb B4 signaling pathway in skin wound repair.We combined multiple research tools of molecular biology,genetics,morphology and pharmacology to explore the expression pattern,activation,and regulation of this signaling from a new perspective of neural-skin interaction.Methods:（1）Immunofluorescence staining was used to observe the expression pattern and spatial distribution of Erb B4 in normal and wounded skin from both human beings and mouse,respectively.Keratin 14（Krt14）was used as a specific marker of epidermal stem cells for analysis,Nestinwas used as a specific marker of multipotent hair follicle stem cells.（2）We established an animal model of full-thickness wounds on the back skin of mice and analyzed the changes of NRG1/Erb B4 signaling pathway in m RNA expression,protein levels and kinase activity,both in DRG and skin,at distinct time points after wound healing by Western blot,RT-PCR,and real-time PCR,and so on.（3）To observed the NRG1 gene transcriptional changes after skin injury,we used a mouse model of skin denervation to observe m RNA and protein expression pattern of NRG1 in DRG,peripheral nerves and the skin tissue during wound repairing.（4）By a strategy of non-viral cutaneous gene transfer DNA using in vivo electroporation,we constructed mouse NRG1 plasmids successfully and overexpressed NRG1 protein in the skin tissue,and then analyzed the effect on the skin wound healing.（5）To further explore the role of Erb B4 inwoundrepair,specific inhibitors AG1478 and AG879 was used by pharmacological strategies on full-thickness wounds in C57 mice to inhibit the corresponding Erb B signaling pathway in the skin,and their effects on skin wound healing was observed.（6）We produced heterozygous Erb B4-knockout(Erb B4^+/-)mice and their wild-type littermates to develop full-thickness skin wound model of mice,and observed the difference between them on skin wound healing.（7）By Cre/Loxp-mediated gene-c KO strategy,we developed multiple lines of Erb B4-c KO,such as Kirt14-Cre;Erb B4^-/-mice and Nestin-Cre ER;Nestin-GF-P;Erb B4^-/-mice that selectively deleted Erb B4 gene in distinct cell types of the skin,to explore the specific cellular mechanisms underlying the effect of Erb B4 signaling on skin wound healing.（8）Research tools combined by MS and WB,et al,was used to explore the downstream signaling pathway key molecules,including AKT,CREB,ERK,et al.,that mediate the effect of Erb B4 signaling in skin of Erb B4^-/-and Kirt14-Cre;Erb B4^-/-mice,to provide more insights into the molecular mechanisms of wound repair.results:（1）The expression and activation of Erb B4 tyrosine receptors is induced by wound injury in skin tissues of patients and animals.Immunofluorescence staining showed that Erb B4 expression can be detected in normal skin and wound skin tissue both in human beings and mice.This expression predominately exits in Krt14-positive epidermal stem cells,it also was detected in Nestin-positive hair follicle stem cells,providing potential role of Erb B4 in wound repair.Around the skin wound margin,the number of cells expressing Erb B4 was markedly increased.RT-PCR results showed that Erb B4 expression in the wound margin was significantly upregulated after wound healing.Moreover,Western blotting showed that both the expression of NRG1 protein and the phosphorylation activity of Erb B4 receptors was robustly induced by the wounds even from the first day after skin injury,in a time-dependent manner.These results suggested that NRG1/Erb B4 signaling is responsive to skin injury in the tissue around the wounds.（2）The gene expression of NRG1 was upregulated in DRGs and accumu-lated in peripheral nerves and skin tissue after wound healing.Both results from RT-PCR and semi-quantative real-time PCR showed that m RNA levels of type I/II NRG1 in peripheral neurons was significantly upregu-lated after skin trauma.In consistence with this,Western blot results showed that the expression levels of NRG1 protein was significantly induced by the skin injury.Moreover,we found that the expression of skin NRG1 was markedly ablated in the skin after denervation manipulation,to support the idea that upregulated NRG1 protein in skin are derived from the DGR neurons.（3）NRG1 plays a potential role in regulating skin wound healing through the Erb B4 signaling pathway.Using non-viral in vivo cutaneous electroporation,we found that overexpressing NRG1 protein in the skin and wound margin can promoted skin wound healing.When we use AG1478 at a dose of 500μM to specifically inhibit the phosph-orylation activity of Erb B4 in skin,and found significantly delayed healing of skin wounds on the back of the treated animals.In contrast,an Erb B2receptor inhibitor AG 879 had no significant effect on skin wound healing.To overcome the possible side effect of pharmacological manipulation,we then used Erb B4 knockout mice in next step.The heterozygous KO mice survived normally,but exhibited slowed healing speed of the skin wound from the 3^rd to 7^th days after skin trauma,compared with their wild-type control littermates.（4）The possible cellular and molecular mechanisms of Erb B4 effect on wound healing.Using conditional knockout of Erb B4 in distinct cell types of the skin,we dissected out the cellular target of Erb B4 effect during wound repair.When we deleted Erb B4 protein in Krt14-positive epidermal stem cells,the wound healing was largely delayed after the skin injury in Kirt14-Cre;Erb B4^-/-mice,but that was not observed in Nestin-Cre ER;Nestin-GFP;Erb B4^-/-mice.Thus,these findings provide a specific cellular targets,Krt14-positive epidermal stem cells,to mediate the effect of NRG1/Erb B4 on wound healing.We further found that phosphorylation activity of AKT increased significantly after skin trauma,but was blocked by Erb B4 knocking out,suggesting a potential downstream pathway of Erb B4 effect in wound repair.Conclusion:（1）The expression of NRG1 and phosphorylation activity of Erb B4tyrosine receptors is induced by wound injury in skin tissues,underscoring an interaction between peripheral nerves and skin tissue.（2）Peripheral nerve can regulate skin wound healing through NRG1/Erb B4signaling pathway.Neuronal NRG1 plays may an important regulatory role in skin wound healing through cutaneous Erb B4 receptors.（3）Krt14-positive epidermal stem cells is a specific cellular targets to mediate the effect of NRG1/Erb B4 on wound healing,possibly through downst-ream molecular signals including AKT pathway.In summary,we found there that skin injury upregulated NRG1 signaling promotes wound healing by mainly activating Erb B4 phosphorylation in Krt14-positive epidermal stem cells.This is the first evidence to demonstrate the effect of NRG1/Erb B4 signaling on skin regeneration and wound healing,providing a novel key intrinsic regulatory factor underlying the repair of skin injury.Our findings will greatly help to understand the molecular mechanisms of wound repair,and underscore the link between neuro-modulation and skin injuryprovi-ding novel insights into the neuro-skin regulation new potential therapeutic targets for the treatment of skin wound healing.