The Effect Of P311 On Skin Wound Reepithelialization Or Renal Fibrosis And The Underlying Mechanisms

Part One: The effect of P311 on skin wound reepithelialization and the underlying mechanisms.Background: Wound healing is a dynamic and coordinated process, which involves the collaborative efforts of multiple types and lineages of cells, the extracellular matrix(ECM) and a diverse set of growth factors and cytokines. The failture of wound healing has become an important clinical problem not only in surgery, burn or trauma but also in patients with chronic conditions, such as varicosity and diabetes. Dysfunctional healing often causes lifelong disability, which imposes enormous socioeconomic burdens on the affercted individuals, families and societies. Demographically, the number of patients suffering from impaired healing conditions and chronic wounds is reaching epidemic proportions worldwide. Hence, there is a strong medical and social need to find the multitude of possible mechanisms in the wound healing and to improve the therapeutic approaches enhancing the endogenous tissue regenerative capacity.The normal mammalian responses to skin wound consists of four overlapping but distinct stages: coagulation, inflammation, new tissue formation, and remodeling. New tissue formation starts with the migration of keratinocytes over the injured dermis. Epidermal stem cells(ESC) are located in three distinct niches at least: the bulge region of hair follicles, the basal layer of the epidermis(IFE) and the base of the sebaceous glands, which play an important role in maintaining homeostasis and wound reepithelialization. After injury, the wound stimulates ESC produce more proliferative transit-amplifying cells through their self-renewal activity, wheres these cells contribute to tissue repair by proliferation and differentiation. ESC have become clinical candidates for treating chronic wounds and ulcers.The P311 gene has been mapped to the chromosome 5 in humans and the chromosome 18 in mice, and is primarily expressed in the mouse embryonic brain and persists at a high level in the cerebellum, hippocampus and olfactory bulb during adulthood. The P311 gene encodes an 8-k Da intracellular protein that contains 68 amino acids and does not belong to any known protein family. The protein’s N-terminus contains a PEST domain(rich in Pro, Glu, Ser and Thr), which is highly conserved among humans, mice and chickens. P311 functions in nerve and lung regeneration, glioblastoma invasion, blood pressure homeostasis, myofibroblast differentiation and amoeboid-like migration. We previously found that P311 promoted early hypertrophic scars formation by enhancing the expression of transforming growth factor β1(TGF-β1). However, the possible role of P311 in skin wounds reepithelialization was still unclear? In this study, we investigated the potential roles of P311 in wound reepithelialization using P311 knockout mice and human skin wound tissue. Therefore, current study will focus on:1. Detection of P311 expression and distribution in cutaneous wounds.2. Determination of the effect of P311 on ESC migration.3. Observation of the underlying molecular mechanisms of P311 on ESC migration.Results:1. P311 was upregulated in human and mouse neonatal epidermis.We investigated 5 samples of human cutaneous wounds from biopsy specimens and normal skin tissue from the same subjects, and built the mouse excisional wound splinting model to examine the expression of P311 protein using immunohistochemical analysis. We found that P311 protein were significantly increased in the cytoplasm of neonatal epidermis cells in human and mouse skin wound tissues. Normal human and mouse epidermis cells indicated were negative for P311.2. P311 promoted ESC migration function.Human ESC were isolated and cultured with the method of rapid adhesion to collagenⅣfrom the foreskin of children. The cells were identified using immunofluorescence staining and flow cytometric analysis to detect ESC markers. Immunofluorescence staining showed that cytokeratin 19 and β1 integrin were highly expressed in the cultured cells. Flow cytometric analysis showed that the cultured cells had a high level of α6 integrin and a low level of CD71. The migrating effects of transfected of P311 and its empty control adenovirus into the ESC were examined by a scratch-wound healing assay in vitro. The assay revealed that the distance and the number of migrating cells were significantly increased in the P311-transfected group compared with the control group at the indicated time point.Murine ESC were isolated and cultured from the neonatal mouse and identified a high level of α6 integrin expression and a low level of CD71 expression by flow cytometric analysis. The effects of P311 konckout in ESC migration were examined by a scratch-wound healing assay in vitro. The assay revealed that the distance and the number of migrating cells were markedly decreased in the P311-/- group compared with the P311+/+ group at the indicated time point.We built a mouse excisional wound splinting model to detect the effects of P311 konckout on wound reepithelialization. We found that the animal model could efficiently prevent skin contraction, and the migration distance of neo-keratinocytes in P311+/+ mice was significantly greater than that in P311-/- mice. P311 deficiency impairs wound reepithelialization.3. The roles of Rho GTPases in the P311 enhancement of ESC migration.To evaluate the responsible mechanisms for the P311-induced enhancement of ESC migration, we analyzed the activity of the small GTPases Rho A, Rac1 and Cdc42 using a pulldown assay. Highly expressed P311 could enhance the activitions of the small GTPases Rho A, Rac1 and Cdc42 in cultured human ESC. While the small GTPases Rho A, Rac1 and Cdc42 activitions dramatically decreased in P311-/- ESC than that P311+/+ in mice. Moreover, both a Rho-specific inhibitor and a Rac1 inhibitor, but not a Cdc42 inhibitor, could significantly suppress P311-induced human ESC migration.In summary, these findings highlight the importance of P311 in the regulation of skin wound reepithelialization. Our work is the first to show that the P311 gene is involved in skin wound reepithelialization, probably by accelerating ESC migration and modulating Rho A and Rac1 activity. Our studies could provide a potential target gene for wound reepithelializationPart Two:The effect of P311 on renal fibrosis and the underlying mechanisms.Background: Renal fibrosis is considered to be failed kidney healing process that occurs after the initial insults of diverse injuries. Clinically, a considerable proportion of patients with renal fibrosis finally progress to end-stage kidney failture, which needs lifelong hematodialysis or kidney transplantation. Numerous epidemiological studies indicate that the prevalence of patients with end-stage renal faliture is increasing worldwide. As a result, renal fibrosis has become a major public health problem on a global range, which causes enormous socioeconomic burdens in the affected individual, family and society. Despite the enormity of this issue, current therapy for renal fibrosi in the clinical setting are often scarce and ineffective. An approved treatment specifically targeted to renal fibrosis is almost inexistent. On this background, we are looking forward to finding the multitude of possible pathological mechanisms and the underlying molecular signal in the formation of renal fibrosis.Renal fibrosis is irreversible and progressive in chronic kidney disease(CKD) and is characterized by the accumulation of extracellular matrix(ECM) components in the glomeruli and tubular interstitium. Regardless of the initial cause, renal fibrogenesis is a dynamic and converging process in which almost all kidney cell types participate. The activation of matrix-producing cells is considered a central event in renal fibrogenesis, as it involves fibroblasts, tubular epithelial cells, vascular smooth muscle cells and a subset of macrophages. Tubular epithelial cells are the primary effector cells that contribute to the irreversible progression of late-stage renal fibrosis. Tubular epithelial cells undergo the phenotypic process of epithelial-mesenchymal transition(EMT) in microenvironments containing various pro-fibrotic cytokines, particularly transforming growth factor beta(TGF-β). TGF-β is a crucial regulator of EMT that mediates many pathological CKD-associated processes and causes tubular epithelial cells to begin to express vimentin(a fibroblast marker) and α-smooth muscle actin(α-SMA, a myofibroblast marker).P311 is primarily expressed in the mouse embryonic brain and persists at a high level in the cerebellum, hippocampus and olfactory bulb during adulthood. The P311 gene has been mapped to the chromosome 5 in humans and the chromosome 18 in mice, and it encodes an 8-k Da intracellular protein that contains 68 amino acids and does not belong to any known protein family. The protein’s N-terminus contains a PEST domain(rich in Pro, Glu, Ser and Thr), which is highly conserved among humans, mice and chickens. P311 functions in nerve and lung regeneration, glioblastoma invasion, blood pressure homeostasis, myofibroblast differentiation and amoeboid-like migration. In our previous studies, we found that P311 was highly over-expressed in early hypertrophic scars and was involved in the pathogenesis of hypertrophic scars via induction of a myofibroblastic phenotype and functions such as TGF-β1 and α-SMA expression. Our previous findings led us to explore whether P311 plays a role in renal fibrogenesis. Therefore, current study will focus on:1. Detection of P311 expression and distribution in renal fibrosis tissues.2. Determination of the effect of P311 on renal fibrosis.3. Observation of the underlying molecular mechanisms of P311 on renal fibrosis.Results:1. P311 was upregulated in human and mouse renal fibrosis.We investigated 22 human clinical biopsy specimens of renal fibrosis and 2 normal human kidney tissue samples and induced mouse renal interstitial fibrosis by unilateral ureteral obstruction model to examine the expression of P311 protein. Hematoxylin and eosin(HE) staining showed remarkable tubular dilation and atrophy, interstitial expansion and abundant inflammatory cell infiltration, while masson trichrome staining revealed a large amount of interstitial collagen fibril deposition in human and mouse renal fibrosis. P311 protein were significantly increased in the cytoplasm of some tubular epithelial cells in human and mouse renal fibrosis tissues samples, as evidenced by immunohistochemical analysis. Normal human and mouse kidney tissue samples indicated the normal renal tubules and the little interstitial collagen fibril, and were negative for P3112. P311 is involved in tubular epithelial cells EMT to promote renal fibrogenesis.The effects of P311 konckout into renal fibrosis were examined by means of masson trichrome staining, Real-Time PCR and western blotting. We firstly observed that the interstitial collagen deposition, collagen Ⅰ m RNA and vimentin protein levels were significantly decreased in obstructed kidneys from P311-/- mice compared to P311+/+ mice.As α-SMA was a characteristic marker of myofibroblast activation in organ fibrosis, we detected the α-SMA m RNA and protein expression by Real-Time PCR, immunohistochemical analysis and western bloting. Immunohistochemical analysis revealed that α-SMA protein localized to the cytoplasm of some tubular epithelial cells, and co-localized with P311 positive cells. α-SMA m RNA and protein expression was significantly higher in obstructed kidneys from P311+/+ mice compared to P311-/- mice.3. P311 deficiency down-regulated TGF-β/Smad signaling in renal fibrosis.TGF-β1 is the key cytokine to tubular epithelial cells EMT and is produced in large quantities by injured tubular epithelial cells and macrophages during renal fibrogenesis. We detected the TGF-β1 expression and macrophage populations by immunohistochemical analysis, Real-Time PCR and western bloting. Immunohistochemical analysis demonstrated that TGF-β1 protein localized to the cytoplasm of some tubular epithelial cells, and co-localized with P311 and α-SMA positive cells. TGF-β1 protein expression was significantly higher in obstructed kidneys from P311+/+ mice compared to P311-/- mice, while TGF-β1 m RNA was no difference.As the TGF-β/Smad signaling pathway plays a pivotal role in renal fibrosis pathogenesis, we detected the TGF-β1 receptor and Smad protein expression by Real-Time PCR and western bloting. Real-Time PCR showed that typeⅠTGF-β1 receptor and typeⅡ TGF-β1 receptor m RNA was dramatically lower in P311-/- mice obstructed kidneys compared to P311+/+ mice. Besides P311-/- mice showed obviously impaired p-Smad2, Smad2, p-Smad3, Smad3, Smad4 and Smad7 protein expression in the obstructed kidney compared to P311+/+ mice.In summary, our work shows that the P311 gene is involved in renal fibrogenesis, likely by enhancing TGFβ-induced EMT and modulating TGF-β/Smad signaling. Our findings could provide a novel potential target for the control of renal fibrosis progression in the future.