IRF7: role and regulation in immunity and autoimmunity.

Interferon regulatory factor (IRF) 7 was originally identified as master transcriptional factor that produced IFN-I and regulated innate immune response, subsequent studies have revealed that IRF7 performs a multifaceted and versatile functions in multiple biological processes. In this review, we provide a comprehensive overview on the current knowledge of the role of IRF7 in immunity and autoimmunity. We focus on the latest regulatory mechanisms of IRF7 in IFN-I, including signaling pathways, transcription, translation, and post-translational levels, the dimerization and nuclear translocation, and the role of IRF7 in IFN-III and COVID-19. In addition to antiviral immunity, we also discuss the role and mechanism of IRF7 in autoimmunity, and the further research will expand our understanding of IRF7.

Parallel Genome-Wide CRISPR Screens to Identify State-Dependent Self-Renewal Regulators of Mouse Embryonic Stem Cells.

The pluripotency of embryonic stem cells (ESCs) is more accurately viewed as a continuous developmental process rather than a fixed state. However, the factors that play general or state-specific roles in regulating self-renewal in different pluripotency states remain poorly defined. In this study, parallel genome-wide CRISPR/Cas9 knockout (KO) screens were applied in ESCs cultured in the serum plus LIF (SL) and in the 2i plus LIF (2iL) conditions. The candidate genes were classified into seven groups based on their positive or negative effects on self-renewal, and whether this effect was general or state-specific for ESCs under SL and 2iL culture conditions. We characterized the expression and function of genes in these seven groups. The loss of function of novel pluripotent candidate genes Usp28, Zfp598, and Zfp296 was further evaluated in mouse ESCs. Consistent with our screen, the knockout of Usp28 promotes the proliferation of SL-ESCs and 2iL-ESCs, whereas Zfp598 is indispensable for the self-renewal of ESCs under both culture conditions. The cell phenotypes of Zfp296 KO ESCs under SL and 2iL culture conditions were different. Our work provided a valuable resource for dissecting the molecular regulation of ESC self-renewal in different pluripotency states.

Human umbilical cord mesenchymal stem cells ameliorate acute graft-versus-host disease by elevating phytosphingosine.

Acute graft-versus-host disease (aGVHD) is a prominent barrier to allogeneic hematopoietic stem cell transplantation (allo-HSCT) and even leads to death after HSCT. Human umbilical cord mesenchymal stem cells (HUCMSCs) are effective in aGVHD treatment and have mild side effects, but the underlying mechanisms remain unclear. Phytosphingosine (PHS) is known to prevent the loss of moisture from the skin; regulate epidermal cell growth, differentiation, and apoptosis; and exert bactericidal and anti-inflammatory effects. In this study, our results revealed the efficacy of HUCMSCs in alleviating aGVHD in a murine model, with striking changes in metabolism and significantly elevated PHS levels due to sphingolipid metabolism. In vitro, PHS reduced CD4+ T-cell proliferation, enhanced apoptosis, and reduced T helper 1 (Th1) cell differentiation. Transcriptional analysis of donor CD4+ T cells treated with PHS revealed significant decreases in transcripts regulating proinflammatory pathways, such as nuclear factor (NF)-κB. In vivo, the administration of PHS significantly ameliorated aGVHD development. Collectively, these beneficial effects indicate proof of concept that sphingolipid metabolites could be a safe and effective means to prevent aGVHD in the clinic.

Connexin 43-modified bone marrow stromal cells reverse the imatinib resistance of K562 cells via Ca 2+ -dependent gap junction intercellular communication.

BACKGROUND: Imatinib mesylate (IM) resistance is an emerging problem for chronic myeloid leukemia (CML). Previous studies found that connexin 43 (Cx43) deficiency in the hematopoietic microenvironment (HM) protects minimal residual disease (MRD), but the mechanism remains unknown. METHODS: Immunohistochemistry assays were employed to compare the expression of Cx43 and hypoxia-inducible factor 1α (HIF-1α) in bone marrow (BM) biopsies of CML patients and healthy donors. A coculture system of K562 cells and several Cx43-modified bone marrow stromal cells (BMSCs) was established under IM treatment. Proliferation, cell cycle, apoptosis, and other indicators of K562 cells in different groups were detected to investigate the function and possible mechanism of Cx43. We assessed the Ca 2+ -related pathway by Western blotting. Tumor-bearing models were also established to validate the causal role of Cx43 in reversing IM resistance. RESULTS: Low levels of Cx43 in BMs were observed in CML patients, and Cx43 expression was negatively correlated with HIF-1α. We also observed that K562 cells cocultured with BMSCs transfected with adenovirus-short hairpin RNA of Cx43 (BMSCs-shCx43) had a lower apoptosis rate and that their cell cycle was blocked in G0/G1 phase, while the result was the opposite in the Cx43-overexpression setting. Cx43 mediates gap junction intercellular communication (GJIC) through direct contact, and Ca 2+ is the key factor mediating the downstream apoptotic pathway. In animal experiments, mice bearing K562, and BMSCs-Cx43 had the smallest tumor volume and spleen, which was consistent with the in vitro experiments. CONCLUSIONS: Cx43 deficiency exists in CML patients, promoting the generation of MRD and inducing drug resistance. Enhancing Cx43 expression and GJIC function in the HM may be a novel strategy to reverse drug resistance and promote IM efficacy.

Newly Found Peacekeeper: Potential of CD8+ Tregs for Graft-Versus-Host Disease.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the most effective and potentially curative treatment for a variety of hematologic malignancies. However, graft-versus-host disease (GVHD) is a major obstacle that limits wide application of allo-HSCT, despite the development of prophylactic strategies. Owing to experimental and clinical advances in the field, GVHD is characterized by disruption of the balance between effector and regulatory immune cells, resulting in higher inflammatory cytokine levels. A reduction in regulatory T cells (Tregs) has been associated with limiting recalibration of inflammatory overaction and maintaining immune tolerance. Moreover, accumulating evidence suggests that immunoregulation may be useful for preventing GVHD. As opposed to CD4+ Tregs, the CD8+ Tregs population, which constitutes an important proportion of all Tregs, efficiently attenuates GVHD while sparing graft-versus-leukemic (GVL) effects. CD8+ Tregs may provide another form of cellular therapy for preventing GVHD and preserving GVL effects, and understanding the underlying mechanisms that different from those of CD4+ Tregs is significant. In this review, we summarize preclinical experiments that have demonstrated the role of CD8+ Tregs during GVHD and attempted to obtain optimized CD8+ Tregs. Notably, although optimized CD8+ Tregs have obvious advantages, more exploration is needed to determine how to apply them in the clinic.

Umbilical cord-derived mesenchymal stem cells promote myeloid-derived suppressor cell enrichment by secreting CXCL1 to prevent graft-versus-host disease after hematopoietic stem cell transplantation.

BACKGROUND AIMS: Human mesenchymal stem cells (MSCs) from various tissues have emerged as attractive candidates for the prevention and treatment of graft-versus-host disease (GVHD). However, the molecular machinery that defines and channels the behavior of these cells remains poorly understood. METHODS: In this study, the authors compared the efficacy of four tissue-derived MSC types in controlling GVHD in a murine model and investigated their immunomodulatory effects. RESULTS: Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) effectively decreased the incidence and severity of GVHD, which was mediated by the enrichment of myeloid-derived suppressor cells in GVHD target tissues. RNA sequencing results showed that hUCMSCs highly expressed CXCL1. CONCLUSIONS: These results suggest a novel prophylactic application of hUCMSCs for controlling GVHD after allogeneic hematopoietic stem cell transplantation.

Interplay Between the Intestinal Microbiota and Acute Graft-Versus-Host Disease: Experimental Evidence and Clinical Significance.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for many hematological disorders and autoimmune diseases, but acute graft-versus-host disease (aGVHD) has remained a major obstacle that limits allo-HSCT and exhibits a daunting mortality rate. The gastrointestinal system is among the most common sites affected by aGVHD. Experimental advances in the field of intestinal microbiota research enhanced our understanding - not only of the quantity and diversity of intestinal microbiota - but also their association with homeostasis of the immune system and disease pathogenesis, including that of aGVHD. Meanwhile, ever-growing clinical evidence suggest that the intestinal microbiota is dysregulated in patients who develop aGVHD and that the imbalance may affect clinical outcomes, indicating a potential predictive role for microbiota dysregulation in aGVHD severity and prognosis. The current animal and human studies investigating the intestinal microbiota in aGVHD and the understanding of the influence and management of the microbiota in the clinic are reviewed herein. Taken together, monitoring and remodeling the intestinal microecology following allo-HSCT may provide us with promising avenues for diagnosing, preventing or treating aGVHD in the clinic.

A panel of 4 biomarkers for the early diagnosis and therapeutic efficacy of aGVHD.

BACKGROUNDCytokine biomarkers have already been used to predict acute graft-versus-host disease (aGVHD) onset, nonrelapse mortality, and overall survival in human and mouse models, but the consistency of the consequences between patients and mice has not been evaluated. Furthermore, no study about any biomarker or biomarker panel for aGVHD grading or steroid sensitivity of aGVHD patients simultaneously has been reported.METHODSHere we established an aGVHD mouse model and explored the relation between aGVHD onset and variations of some cytokines. Based on the results and latest progress, we selected 16 cytokines and compared their serum variations in aGVHD patients and non-aGVHD patients after allogeneic hematopoietic stem cell transplantation. Using protein microarray, we explored the relation between the cytokine levels and aGVHD-related events (onset, grading, and steroid sensitivity).RESULTSThe increase of chemokine levels in murine aGVHD was very consistent with that of patients. We found obviously higher levels of IL-2, IL-4, Elafin, sST2, TLR4, and TNF-α, and lower levels of TGF-ß in both aGVHD mouse models and aGVHD patients. In addition, patients with severe aGVHD showed increased IL-6, TLR4, TNF receptor 1 (TNFR1), and Elafin and decreased TGF-ß. TLR4 and TNFR1 were significantly increased in steroid-refractory aGVHD patients compared with steroid-effective patients (P < 0.05).CONCLUSIONA combination of TLR4, TNFR1, TGF-ß, and Elafin could be a new 4-biomarker panel to assist aGVHD diagnosis, grading, and evaluation of steroid sensitivity for clinical aGVHD patients.TRIAL REGISTRATIONChiCTR1900022292 "Clinical Research of Umbilical Cord-Derived Mesenchymal Stromal Cells in the Prophylaxis of Graft-Versus-Host Disease After HLA-Haploidentical Stem-Cell Transplantation."FUNDINGNational Key Research Program, National Natural Science Foundation of China, Chongqing Social Career and People's Livelihood Security Science and Technology Innovation Project, Fundamental and Frontier Research Program of Chongqing, and Foundation of Xinqiao Hospital.

Developing role of B cells in the pathogenesis and treatment of chronic GVHD.

Chronic graft-versus-host disease (cGVHD) is a major complication affecting the long-term survival of patients after allogeneic haematopoietic stem cell transplantation. The mechanism of cGVHD is unclear, and while previous studies have primarily focused on T cells, the role of B cells in the pathogenesis of cGVHD has been less reported. However, current studies on cGVHD are increasingly focused on the important role of B cells. In this review, we will introduce the newest studies and examine the role of B cells in cGVHD in detail with respect to the following aspects: altered B cell subpopulations, aberrant B cell signalling pathways, autoantibodies and T-B cell interactions. Treatment strategies for the targeting of B cells during cGVHD will also be discussed.

Integrin ß3 Mediates the Endothelial-to-Mesenchymal Transition via the Notch Pathway.

BACKGROUND/AIMS: Neointimal hyperplasia is responsible for stenosis, which requires corrective vascular surgery, and is also a major morphological feature of many cardiovascular diseases. This hyperplasia involves the endothelial-to-mesenchymal transition (EndMT). We investigated whether integrin ß3 can modulate the EndMT, as well as its underlying mechanism. METHODS: Integrin ß3 was overexpressed or knocked down in human umbilical vein endothelial cells (HUVECs). The expression of endothelial markers and mesenchymal markers was determined by real-time reverse transcription PCR (RT-PCR), immunofluorescence staining, and western blot analysis. Notch signaling pathway components were detected by real-time RT-PCR and western blot analysis. Cell mobility was evaluated by wound-healing, Transwell, and spreading assays. Fibroblast-specific protein 1 (FSP-1) promoter activity was determined by luciferase assay. RESULTS: Transforming growth factor (TGF)-ß1 treatment or integrin ß3 overexpression significantly promoted the EndMT by downregulating VE-cadherin and CD31 and upregulating smooth muscle actin α and FSP-1 in HUVECs, and by enhancing cell migration. Knockdown of integrin ß3 reversed these effects. Notch signaling was activated after TGF-ß1 treatment of HUVECs. Knockdown of integrin ß3 suppressed TGF-ß1-induced Notch activation and expression of the Notch downstream target FSP-1. CONCLUSION: Integrin ß3 may promote the EndMT in HUVECs through activation of the Notch signaling pathway.

B7-H3 Promotes the Migration and Invasion of Human Bladder Cancer Cells via the PI3K/Akt/STAT3 Signaling Pathway.

Bladder cancer is one of most common malignant cancer. Although previous studies have found abnormal expression of B7-H3 in human bladder cancer tissues, the exact role and molecular mechanism of B7-H3 in bladder cancer remain unknown. In this study, we first detected the expression of B7-H3 in human bladder cancer samples and cell lines, and analyzed its correlations with clinicopathological pathological parameters. Next, siRNAs or overexpression plasmids of B7-H3 were transfected into T24 or 5637 cells, and cell proliferation, apoptosis, migration and invasion were analyzed via CCK-8, colony formation, flow cytometry and transwell assays, protein expression levels were determined by western blotting. The results presented here showed B7-H3 was upregulated in bladder cancer samples compared with normal tissues, and the expression level was correlated with local invasion status. B7-H3 did not affect cell proliferation and apoptosis, but cell migration and invasion were changed through the regulation of matrix metalloproteinase (MMP) 2/9. Knockdown of B7-H3 resulted in decreased activity of the STAT3 and PI3K/Akt pathways, and the Akt served as an upstream regulator of the STAT3. Our results suggest that the overexpression of B7-H3 promotes the migration and invasion of human bladder cancer cells through the PI3K/Akt/STAT3 signaling pathway.

Blockade of CD47 ameliorates autoimmune inflammation in CNS by suppressing IL-1-triggered infiltration of pathogenic Th17 cells.

The migration of Th17 cells into central nervous system (CNS) tissue is the key pathogenic step in experimental autoimmune encephalomyelitis (EAE) model. However, the mechanism underlying the pathogenic Th17 cell migration remains elusive. Here we report that blockade of CD47 with CD47-Fc fusion protein is effective in preventing and curing EAE by impairing infiltration of Th17 cells into CNS. However, CD47 deficiency does not directly impair the migration of Th17 cells. Mechanistic studies showed that CD47 deficiency inhibited degradation of inducible nitric oxide synthase (iNOS) in proteasome of macrophages by Src activation and led to the increased nitric oxide (NO) production. Then NO suppressed inflammasome activation-induced IL-1ß production. This lower IL-1ß reduces the expression of IL-1R1 and migration-related chemokine receptors on CD47(-/-) Th17 cells, inhibiting the ability of Th17 cells to infiltrate into the CNS of CD47(-/-) mice and therefore suppressing EAE development. In vivo administration of exogenous IL-1ß indeed promoted the infiltration CD47(-/-) Th17 cells into CNS and antagonized the protective role of CD47 deficiency in EAE pathogenesis. Our results demonstrate a potential preventive and therapeutic application of CD47 blockade in controlling EAE development.

[Experimental study on the effects of the nerve growth factor regulating calcitonin gene-related peptide in promoting the proliferation of MG-63 in vitro].

OBJECTIVE: To investigate the nerve growth factor (NGF) regulating the expression of calcitonin gene-related peptide (CGRP) in promoting the proliferation of osteoblast-like cell (MG-63) and thus illustrate the mechanism of the NGF in wound healing. METHODS: Different concentrations of NGF were used to stimulate MG-63. The expression of CGRP was detected by real-time quantitative polymerase chain reaction (RT-QPCR) and enzyme-linked immunosorbent assay after 1, 2, 3, and 4 days. The proliferation of MG-63 was detected by cell counting kit-8 (CCK-8). The expression of CGRP mRNA and the proliferation of MG-63 were then detected by RT-QPCR and CCK-8 after adding the NGF receptor blocker. RESULTS: Compared with the blank control group, the expression of CGRP significantly increased by stimulating the NGF. The expression of CGRP was positively related to the concentration of NGF (P<0.05). Moreover, the expression of CGRP increased by prolonging the NGF stimulation time. The proliferation of MG-63 increased after stimulating the NGF (P<0.05). After adding the NGF receptor blocker, the expression of CGRP and the proliferation of MG-63 correspondingly decreased (P<0.05). CONCLUSION: NGF can up-regulate the expression of CGRP and increase the proliferation of MG-63. Therefore, NGF plays a significant role in wound healing.

A novel siRNA validation system for functional screening of effective RNAi targets in mammalian cells and development of a derivative lentivirus delivery system.

RNA interference technology is a widely used tool for the regulation of gene expression at the post-transcriptional level. One major challenge is to find the effective short interfering (si)RNA for target gene rapidly and easily, and then to deliver the siRNA into cells or tissues with high efficiency. Here, we designed a novel siRNA validation vector using a dual luciferase reporter system for the functional screening of effective RNAi targets in mammalian cells. Then, based on a siRNA expression cassette, we developed a derivative lentivirus delivery system to infect the appropriate cells or tissues for the efficient knockdown of target gene expression. Based on this system, we used human IRF7 gene, a key regulatory factor for the differentiation of monocytes to macrophages, as an example. We screened for the optimal siRNA, then packaged it into a lentiviral siRNA expression system. Then, monocytes were infected and we confirmed the knockdown of IRF7 expression could inhibit the differentiation of monocytes to macrophages. To validate our method further, we also screened and identified optimal siRNA for human TLR4 gene. In summary, we developed a novel siRNA validation system to identify optimal siRNA to target genes rapidly. In addition, the lentivirus system is an efficient tool for siRNA delivery for the further study of target gene function. Taken together, this represents an efficient and user-friendly strategy to validate and deliver siRNAs.

NF-κB plays a key role in inducing CD274 expression in human monocytes after lipopolysaccharide treatment.

CD274, one of two co-stimulatory ligands for programmed death 1 and widely expressed in the mononuclear phagocyte system (MPS), may co-stimulate T cells and regulates inflammatory responses. However, changes in CD274 gene expression and the underlying molecular mechanism are poorly understood during inflammatory responses. Therefore, delineation of the complex mechanisms regulating CD274 expression is critical to understand this immunoregulatory system during inflammatory responses. The purpose of this study was to assess the molecular mechanisms regulating CD274 expression in an in vitro monocyte model of inflammatory response. Firstly, CD274 expression levels in human primary monocytes after lipopolysaccharide (LPS) treatment were observed and correlated with NF-κB activation. Secondly, based on the distribution of putative NF-κB binding sites, 5' truncated human CD274 promoter reporters were constructed, transfected into U937 cells and critical promoter regions for basal (nt -570 to +94) and LPS-induced (nt -1735 to -570) transcription were identified by dual luciferase assays. Finally, a key NF-κB binding site (nt -610 to -601) for LPS-inducible CD274 transcriptional activity was characterized by point mutation analysis and chromatin immunoprecipitation analysis assays (ChIP). Thus, the present study establishes a molecular basis to understand the mechanisms governing CD274 expression in certain infections and inflammatory disorders.

An efficient and rapid method for cDNA cloning from difficult templates using codon optimization and SOE-PCR: with human RANK and TIMP2 gene as examples.

As gene cloning from difficult templates with regionalized high GC content is a long recognized problem, we have developed a novel and reliable method to clone such genes. Firstly, the high GC content region of the target cDNA was synthesized directly after codon optimization and the remaining cDNA fragment without high GC content was generated by routine RT-PCR. Then the entire redesigned coding sequence of the target gene was obtained by fusing the above available two cDNA fragments with SOE-PCR (splicing by overlapping extension-PCR). We have cloned the human RANK gene (ten exons; CDS 1851 bp) using this strategy. The redesigned cDNA was transfected into an eukaryotic expression system (A459 cells) to verify its expression. RT-PCR and western blotting confirmed this. To validate our method, we also successfully cloned human TIMP2 gene (five exons; CDS 660 bp) also having a regionalized high GC content. Our strategy for combining codon optimization and SOE-PCR to clone difficult genes is thus feasible and potentially universally applicable.

Regulatory dendritic cells program generation of interleukin-4-producing alternative memory CD4 T cells with suppressive activity.

The heterogeneity and mechanisms for the generation of CD4 memory T (CD4 Tm) cells remain elusive. Distinct subsets of dendritic cells (DCs) have been found to regulate a distinct T-helper (Th)-cell subset differentiation by influencing cytokine cues around CD4 T cells; however, whether and how the regulatory DC subset can regulate Tm-cell differentiation remains unknown. Further, there is no ideal in vitro experimental system with which to mimic the 3 phases of the CD4 T-cell immune response (expansion, contraction, memory generation) and/or to culture CD4 Tm cells for more than a month. By analyzing CD4 T cells programmed by long-term coculture with regulatory DCs, we identified a population of long-lived CD4 T cells with a CD44(hi)CD62L(-)CCR7(-) effector memory phenotype and rapid, preferential secretion of the Th2 cytokines interleukin-4 (IL-4), IL-5, IL-10, and IL-13 after antigenic stimulation. These regulatory DC-programmed Tm cells suppress CD4 T-cell activation and proliferation in vitro via IL-10 and inhibit the delayed-type hypersensitivity response once infused in vivo. We also identify their natural counterpart, which is up-regulated by regulatory DC transfusion and negatively regulates the recall response in vivo. Different from interferon-γ-producing conventional Tm cells, these IL-4-producing CD4 Tm cells act as alternative Tm cells with a regulatory function, suggesting a new way of negative immune regulation by memory T cells.

Committed differentiation of hair follicle bulge cells into sebocytes: an in vitro study.

BACKGROUND: Several studies have shown that hair follicle bugle cells can differentiate into hair follicles and contribute to the formation of the epidermis and sebaceous gland. Although many lines of evidence have suggested that the renewal and maintenance of the sebaceous gland depends on hair follicle bulge cells, direct evidence supporting the in vitro differentiation of follicle bulge cells into sebaceous gland cells has not been found. METHODS: Rat vibrissa follicle bulge cells were isolated, cultured, and transfected with green fluorescent protein (GFPC1) plasmids carrying the peroxisome proliferator-activated receptor gamma2 (PPARgamma2 ) gene. The transfected cells were cultured in modified medium, and the morphologic changes of the cells were observed. Moreover, the expression of epithelial membrane antigens (EMAs) by the cells was detected by immunocytochemistry, and adipogenesis of the cells was evaluated. RESULTS: After induction culture, the cell body enlarged gradually and contained abundant cytoplasm; lipid droplets appeared in the cytoplasm of some cells, and the cells resembled sebocytes of the sebaceous gland. The cells were positive on oil red O and EMA staining. The expression of PPARgamma2 mRNA and protein was significantly upregulated in PPARgamma2-transfected cells. The rate of oil red O-stained and EMA-positive cells was higher in PPARgamma2-transfected cells after induction than in bulge-PPARgamma2 cells and non-transfected bulge cells. CONCLUSIONS: Rat vibrissa hair follicle bulge cells may differentiate into sebocytes in vitro, and the PPARgamma2 gene plays a crucial role.

Relationship between urokinase plasminogen activator receptor (uPAR) and the invasion of human prenatal hair follicle.

During the morphogenesis of hair follicles, the invasive migration of basal keratinocytes resembles cell's dissemination of tissue remodeling. The urokinase-type plasminogen activator receptor (uPAR) appears to be a key molecule in the metastasis. In order to elucidate the relationship between uPAR and the invasion of the human hair follicle, immunohistochemistry, RT-PCR, plasmids transfection, and western blot were used. The results showed that uPAR was expressed in the outermost epithelial cells of the hair follicle and the basal keratinocytes of epidermis, and that the expression decreased with the development of the hair follicle. The cells of the outer root sheath (ORS) and interfollicle epidermis, which overexpressed uPAR, acquired increased invasiveness; however, they showed decreased invasion with overexpression of the urokinase-type plasminogen activator amino terminal fragment (uPA ATF), which inhibited the combination of uPAR and uPA competitively, and the cell invasive migration with overexpressed uPAR was required activated extracellular signal-regulated kinases (ERK). These results implied that overexpression of uPAR promote the invasive migration of hair follicle into the dermis in uPA-dependent and independent manner during human prenatal development.

IL-1alpha is a potent stimulator of keratinocyte tissue plasminogen activator expression and regulated by TGF-beta1.

Epidermal keratinocytes synthesize and secrete tissue-type plasminogen activator. tPA converts the inactive precursor enzyme plasminogen into the trypsin like proteinase plasmin, and is an essential component of normal wound healing. tPA is not found in normal epidermis, but in keratinocytes at the leading edge of the wound. Proteolytic activity produced by tPA is necessary for the wound epidermis to cleave fibrin from its migratory pathway. However, the factors that regulate tPA expression in keratinocytes have remained largely elusive. In the present study, we sought to determine whether the cytokine interleukin-1alpha (IL-1alpha) and its regulator transforming growth factor beta1 (TGF-beta1), which are both commonly present in skin wounds, influence tPA production in cultured murine epidermal keratinocytes. We treated the keratinocytes with IL-1alpha in the absence or presence of TGF-beta1 at various concentrations and analyzed the expression of tPA by RT-PCR, in situ hybridization, immunocytochemistry as well as immunofluorescence. We found that IL-1alpha induced mRNA and protein expression of tPA in a time and dose dependent manners. The maximal stimulation was seen at 72 h and 100 U/ml. However, treatment of keratinocytes with TGF-beta1 partially inhibited IL-1alpha induced expression of tPA mRNA in dose dependent manners and the maximal inhibition was seen at 60 ng/ml. Our findings could explain tPA generation in wound epidermis is at least partially controlled by changes in local IL-1alpha activity, and will contribute to our understanding the physiological effects of IL-1alpha and TGF-beta1 as well as their interaction of with the PA system during skin wound healing.