Association of suPAR, ST2, and galectin-3 with eGFR decline and mortality in patients with advanced heart failure with reduced ejection fraction.

Patients with heart failure with reduced ejection fraction (HFrEF) are at risk for chronic kidney disease (CKD). Elevated levels of circulating biomarkers soluble urokinase plasminogen activator receptor (suPAR), galectin-3, soluble suppression of tumorigenicity 2 (ST2), and N-terminal prohormone B-type natriuretic peptide (NT-proBNP) are associated with CKD progression and mortality. The predictive value of these biomarkers in a population with HFrEF and kidney disease is relatively unknown. We sought to determine whether these biomarkers were associated with longitudinal trajectory of estimated glomerular filtration rate (eGFR) in HFrEF and assess their association with mortality using a joint model to account for competing risks of ventricular assist device (VAD) implantation and heart transplantation. We included participants from the Registry Evaluation of Vital Information for Ventricular Assist Devices in Ambulatory Life with repeated eGFR measures over 2 years. Of 309 participants, mean age was 59 years, median eGFR 60 ml/min/1.73 m2, 45 participants died, 33 received VAD, and 25 received orthotopic heart transplantation. Higher baseline serum standardized suPAR (ß coefficient = -0.36 √(ml/min/1.73 m2), 95% confidence interval (-0.48 to -0.24), p < 0.001), standardized galectin-3 (-0.14 √(ml/min/1.73 m2) (-0.27 to -0.02), p = 0.02), and log NT-proBNP (-0.23 √(ml/min/1.73 m2) (-0.31 to -0.15), p < 0.001) were associated with eGFR decline. ST2 and log NT-proBNP were associated with mortality. Higher baseline suPAR, galectin-3, and NT-proBNP are associated with eGFR decline in patients with HFrEF. Only ST2 and NT-proBNP are associated with greater mortality after controlling for other factors including change in eGFR. These biomarkers may provide prognostic value for kidney disease progression in HFrEF and inform candidacy for advanced heart failure therapies.

Acute hepatic and kidney injury after ingestion of Lepiota brunneoincarnata: Report of 2 cases.

Lepiota brunneoincarnata is a highly toxic mushroom species known to cause acute liver failure. However, there are limited reports investigating L. brunneoincarnata causing acute hepatic and renal damage. The present article reports 2 cases of L. brunneoincarnata poisoning in a mother and son from Chuxiong City, Yunnan Province, China. Both patients presented with gastrointestinal symptoms approximately 8-9 h after ingesting the suspect mushrooms and sought medical attention 27-28 h post-ingestion, both exhibiting acute hepatic and kidney injuries. Morphological and molecular biology studies confirmed the species of the mushrooms as L. brunneoincarnata. Liquid chromatography-tandem mass spectrometry analysis revealed mean fresh-weight concentrations of 123.5 µg/g α-amanitin and 45.7 µg/g ß-amanitin in the mushrooms. The patients underwent standard treatments, including multiple-dose activated charcoal, oral silibinin capsules, N-acetylcysteine, penicillin G, hemoperfusion, and plasma exchange. One patient recovered completely and was discharged after 16 days of hospitalization. The other patient exhibited gradual improvement in liver and renal function; however, renal function deteriorated 9 days after ingestion, and the patient declined renal replacement therapy and returned home 14 days post-ingestion. The patient was then re-hospitalized due to oliguria and edema in both lower extremities. Renal biopsy revealed acute tubular necrosis, inflammatory cell infiltration, minor glomerular capsular fibrosis, loss of microvilli in the renal tubular epithelial cells, and interstitial edema. The patient underwent 2 rounds of continuous renal replacement therapy, which eventually resulted in improvement, and was discharged 31 days after mushroom consumption. It is noteworthy that this patient had already progressed to chronic kidney insufficiency 11 months after intoxication.

Smarca4 deficiency induces Pttg1 oncogene upregulation and hyperproliferation of tubular and interstitial cells during kidney development.

Kidney formation and nephrogenesis are controlled by precise spatiotemporal gene expression programs, which are coordinately regulated by cell-cycle, cell type-specific transcription factors and epigenetic/chromatin regulators. However, the roles of epigenetic/chromatin regulators in kidney development and disease remain poorly understood. In this study, we investigated the impact of deleting the chromatin remodeling factor Smarca4 (Brg1), a human Wilms tumor-associated gene, in Wnt4-expressing cells. Smarca4 deficiency led to severe tubular defects and a shortened medulla. Through unbiased single-cell RNA sequencing analyses, we identified multiple types of Wnt4 Cre-labeled interstitial cells, along with nephron-related cells. Smarca4 deficiency increased interstitial cells but markedly reduced tubular cells, resulting in cells with mixed identity and elevated expression of cell-cycle regulators and genes associated with extracellular matrix and epithelial-to-mesenchymal transition/fibrosis. We found that Smarca4 loss induced a significant upregulation of the oncogene Pttg1 and hyperproliferation of Wnt4 Cre-labeled cells. These changes in the cellular state could hinder the cellular transition into characteristic tubular structures, eventually leading to fibrosis. In conclusion, our findings shed light on novel cell types and genes associated with Wnt4 Cre-labeled cells and highlight the critical role of Smarca4 in regulating tubular cell differentiation and the expression of the cancer-causing gene Pttg1 in the kidney. These findings may provide valuable insights into potential therapeutic strategies for renal cell carcinoma resulting from SMARCA4 deficiency.

Cardiac myxoma shedding leads to lower extremity arterial embolism: A case report.

BACKGROUND: Left cardiac myxoma (CM) is the most common benign tumor of primary cardiac tumors, but because of its special position caused by pathological physiology change, caused by the complications of the heavier, the surface is often accompanied by blood clots, once fall out, it causes peripheral vascular embolization, such as acute lower limb artery embolization, harmfulness is large, high morbidity, and easy to occur repeatedly. CASE SUMMARY: A 67-year-old male patient suddenly appeared numbness and weakness of the left lower limb and could not walk without obvious incentive. The patient was finally diagnosed as left CM complicated with acute lower limb arterial embolism after completing cardiac ultrasound, computer tomography angiography, and histopathological analysis, such as hematoxylin-eosin stain staining, immunohistochemistry and special staining including alcian blue staining and periodic acid schiff staining. Arterial thrombosis was removed successfully by femoral artery thrombectomy, postoperative numbness and weakness of the patient's left lower limb disappeared, skin temperature became warm, and dorsal foot artery pulsation was accessible. The patient was readmitted to the hospital 8 mo after discharge for left atrial mass resection, and was diagnosed as CM by postoperative histopathological examination. CONCLUSION: Although CM is rare, it may be considered as the source of embolism in patients with acute limb ischemia. Repeated loss of thrombus on the tumor and its surface may lead to repeated embolism of peripheral vessels. Cardiac ultrasound is helpful for early diagnosis. Here, we use this case report to highlight left CM as an important cause of acute limb ischemia and to report our experience in the diagnosis and treatment of lower limb arterial embolism caused by CM detachment.

The transcriptional coactivator Eya1 exerts transcriptional repressive activity by interacting with REST corepressors and REST-binding sequences to maintain nephron progenitor identity.

Eya1 is critical for establishing and maintaining nephron progenitor cells (NPCs). It belongs to a family of proteins called phosphatase-transcriptional activators but without intrinsic DNA-binding activity. However, the spectrum of the Eya1-centered networks is underexplored. Here, we combined transcriptomic, genomic and proteomic approaches to characterize gene regulation by Eya1 in the NPCs. We identified Eya1 target genes, associated cis-regulatory elements and partner proteins. Eya1 preferentially occupies promoter sequences and interacts with general transcription factors (TFs), RNA polymerases, different types of TFs, chromatin-remodeling factors with ATPase or helicase activity, and DNA replication/repair proteins. Intriguingly, we identified REST-binding motifs in 76% of Eya1-occupied sites without H3K27ac-deposition, which were present in many Eya1 target genes upregulated in Eya1-deficient NPCs. Eya1 copurified REST-interacting chromatin-remodeling factors, histone deacetylase/lysine demethylase, and corepressors. Coimmunoprecipitation validated physical interaction between Eya1 and Rest/Hdac1/Cdyl/Hltf in the kidneys. Collectively, our results suggest that through interactions with chromatin-remodeling factors and specialized DNA-binding proteins, Eya1 may modify chromatin structure to facilitate the assembly of regulatory complexes that regulate transcription positively or negatively. These findings provide a mechanistic basis for how Eya1 exerts its activity by forming unique multiprotein complexes in various biological processes to maintain the cellular state of NPCs.

Label-free detection of breast cancer cells using a functionalized tilted fiber grating.

The detection of circulating tumor cells (CTCs) still faces a huge challenge partially because of low abundance of CTCs (1-10 cells/mL). In this work, a plasmonic titled fiber Bragg grating biosensor is proposed for detection of breast cancer cells. The biosensor is made by an 18° TFBG with a 50 nm-thick gold nanofilm coating over the surface of the fiber, further immobilized with a specific antibody against GPR30, which is a membrane receptor expressed in many breast cancers, serving as bait. In vitro tests have confirmed that the proposed biosensor can detect breast cancer cells in concentration of 5 cells/mL within 20 minutes and has good linearity in the range of 5-1000 cells/mL, which has met the requirement of CTC detection in real conditions. Furthermore, theoretical analysis based on the experimental results shows that the limit of detection can even reach single-cell level. Our proposed biosensor has a simple structure, is easy to manufacture, is of small size, and has a good performance, making it a good choice for real-time, label-free, and milliliter-volume detection of cancer cells in future.

Intravascular fasciitis involving the external jugular vein and subclavian vein: A case report.

BACKGROUND: Intravascular fasciitis (IVF) is a rare nodular fasciitis that often involves the layers and lumens of blood vessels; therefore, it is easily misdiagnosed as a malignant tumor with invasion into blood vessels. CASE SUMMARY: A 13-year-old boy was admitted due to a mass on the left side of his neck. Duplex ultrasonography revealed a circular solid hypoechoic mass in the external jugular vein, and magnetic resonance imaging revealed an enhanced longitudinal mass-like lesion in the left supraclavicular fossa. Surgical treatment was arranged and completed, histopathological analysis showed a large amount of spindle cell proliferation, and immunohistochemistry showed that the spindle cells were positive for the expression of vimentin, caldesmon, and smooth muscle actin and negative for the expression of S-100 protein, desmin, CD34, and c-kit; Ki-67 staining revealed a low proliferative index (5%-10%), which confirmed the differentiation characteristics of myofibroblasts. Fluorescence in situ hybridization detected the rearrangement of USP6. IVF was subsequently diagnosed. CONCLUSION: IVF is characterized by intraluminal, intramural and extramural involvement of small to large arteries or veins. Unless the doctor has a deep understanding of the disease or suspects that there is an initial indicator, IVF may be confused with other intravascular malignancies, leading to unnecessary radical surgery. Imaging examination combined with histopathological examination can improve the diagnostic accuracy of this disease.

Chromatin Remodelers Interact with Eya1 and Six2 to Target Enhancers to Control Nephron Progenitor Cell Maintenance.

BACKGROUND: Eya1 is a critical regulator of nephron progenitor cell specification and interacts with Six2 to promote NPC self-renewal. Haploinsufficiency of these genes causes kidney hypoplasia. However, how the Eya1-centered network operates remains unknown. METHODS: We engineered a 2×HA-3×Flag-Eya1 knock-in mouse line and performed coimmunoprecipitation with anti-HA or -Flag to precipitate the multitagged-Eya1 and its associated proteins. Loss-of-function, transcriptome profiling, and genome-wide binding analyses for Eya1's interacting chromatin-remodeling ATPase Brg1 were carried out. We assayed the activity of the cis-regulatory elements co-occupied by Brg1/Six2 in vivo. RESULTS: Eya1 and Six2 interact with the Brg1-based SWI/SNF complex during kidney development. Knockout of Brg1 results in failure of metanephric mesenchyme formation and depletion of nephron progenitors, which has been linked to loss of Eya1 expression. Transcriptional profiling shows conspicuous downregulation of important regulators for nephrogenesis in Brg1-deficient cells, including Lin28, Pbx1, and Dchs1-Fat4 signaling, but upregulation of podocyte lineage, oncogenic, and cell death-inducing genes, many of which Brg1 targets. Genome-wide binding analysis identifies Brg1 occupancy to a distal enhancer of Eya1 that drives nephron progenitor-specific expression. We demonstrate that Brg1 enrichment to two distal intronic enhancers of Pbx1 and a proximal promoter region of Mycn requires Six2 activity and that these Brg1/Six2-bound enhancers govern nephron progenitor-specific expression in response to Six2 activity. CONCLUSIONS: Our results reveal an essential role for Brg1, its downstream pathways, and its interaction with Eya1-Six2 in mediating the fine balance among the self-renewal, differentiation, and survival of nephron progenitors.

NPAS4 regulates the transcriptional response of the suprachiasmatic nucleus to light and circadian behavior.

The suprachiasmatic nucleus (SCN) is the master circadian pacemaker in mammals and is entrained by environmental light. However, the molecular basis of the response of the SCN to light is not fully understood. We used RNA/chromatin immunoprecipitation/single-nucleus sequencing with circadian behavioral assays to identify mouse SCN cell types and explore their responses to light. We identified three peptidergic cell types that responded to light in the SCN: arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), and cholecystokinin (CCK). In each cell type, light-responsive subgroups were enriched for expression of neuronal Per-Arnt-Sim (PAS) domain protein 4 (NPAS4) target genes. Further, mice lacking Npas4 had a longer circadian period under constant conditions, a damped phase response curve to light, and reduced light-induced gene expression in the SCN. Our data indicate that NPAS4 is necessary for normal transcriptional responses to light in the SCN and critical for photic phase-shifting of circadian behavior.

SIX1/EYA1 are novel liver damage biomarkers in chronic hepatitis B and other liver diseases.

BACKGROUND: This study aimed to investigate the clinicopathological significance of sine oculis homeobox homolog 1 (SIX1) and eyes absent 1 (EYA1) in patients with chronic hepatitis B (CHB) and other liver diseases. METHODS: SIX1 and EYA1 levels were detected in human serum and liver tissues by enzyme linked immunosorbent assay (ELISA) and immunofluorescent staining method, respectively. RESULTS: The serum SIX1 and EYA1 levels in 313 CHB patients were 7.24±0.11 and 25.21±0.51 ng/mL, respectively, and these values were significantly higher than those in 33 healthy controls (2.84±0.15 and 13.11±1.01 ng/mL, respectively; P<0.05). Serum SIX1 and EYA1 levels were also markedly increased in patients with numerous other liver diseases, including liver fibrosis, hepatocellular carcinoma, fatty liver disease, alcoholic liver disease, fulminant hepatic failure, autoimmune liver disease, and hepatitis C, compared to the healthy controls (P<0.05). Dynamic observation of these proteins over time in 35 selected CHB patients revealed that SIX1 and EYA1 serum levels increased over an interval. Immunofluorescent staining revealed that both SIX1 and EYA1 were only expressed in hepatic stellate cells (HSCs), and their increased expression was evident in CHB liver tissue. CONCLUSIONS: SIX1 and EYA1 are novel biomarkers of liver damage in patients of CHB and other liver diseases, with potential clinical utility.

The Lysosomal Membrane Protein Lamp2 Alleviates Lysosomal Cell Death by Promoting Autophagic Flux in Ischemic Cardiomyocytes.

Lysosomal membrane permeabilization (LMP) has recently been recognized as an important cell death pathway in various cell types. However, studies regarding the correlation between LMP and cardiomyocyte death are scarce. Lysosomal membrane-associated protein 2 (Lamp2) is an important component of lysosomal membranes and is involved in both autophagy and LMP. In the present study, we found that the protein content of Lamp2 gradually decreased in response to oxygen, glucose and serum deprivation (OGD) treatment in vitro. To further elucidate its role in ischemic cardiomyocytes, particularly with respect to autophagy and LMP, we infected cardiomyocytes with adenovirus carrying full-length Lamp2 to restore its protein level in cells. We found that OGD treatment resulted in the occurrence of LMP and a decline in the viability of cardiomyocytes, which were remarkably reversed by Lamp2 restoration. Exogenous expression of Lamp2 also significantly alleviated the autophagic flux blockade induced by OGD treatment by promoting the trafficking of cathepsin B (Cat B) and cathepsin D (Cat D). Through drug intervention and gene regulation to alleviate and exacerbate autophagic flux blockade respectively, we found that impaired autophagic flux in response to ischemic injury contributed to the occurrence of LMP in cardiomyocytes. In conclusion, our present data suggest that Lamp2 overexpression can improve autophagic flux blockade probably by promoting the trafficking of cathepsins and consequently conferring cardiomyocyte resistance against lysosomal cell death (LCD) that is induced by ischemic injury. These results may indicate a new therapeutic target for ischemic heart damage.

ZBP-89 and Sp1 contribute to Bak expression in hepatocellular carcinoma cells.

BACKGROUND: Kruppel family member zinc binding protein 89 (ZBP-89), also known as ZNF148, regulates Bak expression via binding to GC-rich promoter domain. It is not clear if other GC-rich binding factors, such as Sp family members, can interact with ZBPp-89 on Bak expression. This study aims to elucidate the mechanism of Bak expression regulation by ZBP-89 and Sp proteins, based on in vitro experiment and The Cancer Genome Atlas (TCGA) hepatocellular carcinoma (HCC) data cohort. METHODS: We downloaded TCGA hepatocellular carcinoma (HCC) cohort data to analysis the association of Bak transcription level with ZBP-89 and Sp proteins transcription level. HCC cell lines and liver immortal non-tumour cell lines were used for mechanism study, including western blotting analysis, expression vector mediated gene expression and siRNA interference. RESULTS: Results showed that cancer tissues have higher Bak transcription level compared with adjacent non-cancer tissues. Bak transcription level was correlated with Sp1 and Sp3 expression level, while no correlation was found in ZBP-89 and Bak, neither Sp2 nor Sp4. Mithramycin A (MMA) induced Bak expression in a dose-dependent manner. Western blotting results showed Sp1 overexpression increased Bak expression both in liver immortal non-tumour cells and HCC cells. Interference Sp1 expression could inhibit Bak expression alone. ZBP-89 siRNA suppressed Bak expression even in the presence of MMA treatment and S1 overexpression. Additionally, Bak and Sp1 level were associated with HCC patient survival. CONCLUSIONS: Bak expression required ZBP-89 and Sp1 cooperative regulation simultaneously.

[Inhibitory effect of bispecific antibody targeting IL-12 p40 and TNF-α simultaneously on psoriasis in mice].

Objective To construct bispecific antibodies, which can block interleukin 12 (IL-12)/IL-23 p40 subunit and tumor necrosis factor α (TNF-α) simultaneously, and identify their biological function and inhibitory effect on psoriasis formation in mice. Methods Based on the sequences of adalimumab and ustekinumab, three kinds of bispecific antibodies were designed, named BiAU003, BiAU022 and BiAU023. The specificity and binding capacity of bispecific antibodies were determined by ELISA. After co-treated with bispecific antibodies and TNF-α, the level of endothelial leukocyte adhesion molecule-1 (ELMA-1) labeled with fluorescein isothiocyanate (FITC) in human umbilical vein endothelial cells (HUVECs) were examined by flow cytometry. Human peripheral blood mononuclear cells (PBMCs) were purified and cultured in the medium containing IL-2 and IL-12 in the absence or presence of bispecific antibodies. Commercial ELISA kit was used to detect interferon γ (IFN-γ) concentration in the supernatant. BALB/c mice were used for psoriasis model construction through injection of IL-12 and TNF-α subcutaneously. Then they were treated with the bispecific antibodies. Psoriasic skin was measured in thickness and scale under microscopy after H&E staining. Results The three kinds of bispecific antibodies could specifically recognize IL-12/23 p40 and TNF-α protein, and inhibit IFN-γ secretion and the expression of ELAM-1 protein. Data also indicated that bispecific antibodies inhibited the formation of psoriasic skin, and showed an equal or superior effect to control antibody drug. Conclusion The novel bispecific antibodies, BiAU003, BiAU022 and BiAU023, can serve as an antagonist of TNF-α and IL-12/23 p40, and have a blocking effect on mouse psoriasis formation.

Identification of mouse cochlear progenitors that develop hair and supporting cells in the organ of Corti.

The adult mammalian cochlear sensory epithelium houses two major types of cells, mechanosensory hair cells and underlying supporting cells, and lacks regenerative capacity. Recent evidence indicates that a subset of supporting cells can spontaneously regenerate hair cells after ablation only within the first week postparturition. Here in vivo clonal analysis of mouse inner ear cells during development demonstrates clonal relationship between hair and supporting cells in sensory organs. We report the identification in mouse of a previously unknown population of multipotent stem/progenitor cells that are capable of not only contributing to the hair and supporting cells but also to other cell types, including glia, in cochlea undergoing development, maturation and repair in response to damage. These multipotent progenitors originate from Eya1-expressing otic progenitors. Our findings also provide evidence for detectable regenerative potential in the postnatal cochlea beyond 1 week of age.

EYA1's Conformation Specificity in Dephosphorylating Phosphothreonine in Myc and Its Activity on Myc Stabilization in Breast Cancer.

EYA1 is known to be overexpressed in human breast cancer, in which the Myc protein is also accumulated in association with decreased phospho-T58 (pT58) levels. We have recently reported that EYA1 functions as a unique protein phosphatase to dephosphorylate Myc at pT58 to regulate Myc levels. However, it remains unclear whether EYA1-mediated Myc dephosphorylation on T58 is a critical function in regulating Myc protein stability in breast cancer. Furthermore, EYA1's substrate specificity has remained elusive. In this study, we have investigated these questions, and here, we report that depletion of EYA1 using short hairpin RNA (shRNA) in breast cancer cells destabilizes the Myc protein and increases pT58 levels, leading to an increase in the doubling time and impairment of cell cycle progression. In correlation with EYA1-mediated stabilization of cMyc and reduced levels of pT58, EYA1 greatly reduced cMyc-FBW7 binding and cMyc ubiquitination, thus providing novel insight into how EYA1 acts to regulate the FBW7-mediated Myc degradation machinery. We found that the conserved C-terminal haloacid dehalogenase domain of EYA1, which has been reported to have only tyrosine phosphatase activity, has dual phosphatase activities, and both the N- and C-terminal domains interact with substrates to increase the catalytic activity of EYA1. Enzymatic assay and nuclear magnetic resonance (NMR) analysis demonstrated that EYA1 has a striking conformation preference for phospho-T58 of Myc. Together, our results not only provide novel structural evidence about the conformation specificity of EYA1 in dephosphorylating phosphothreonine in Myc but also reveal an important mechanism contributing to Myc deregulation in human breast cancer.

The Eya1 phosphatase promotes Shh signaling during hindbrain development and oncogenesis.

Sonic hedgehog (Shh) signaling is critical in development and oncogenesis, but the mechanisms regulating this pathway remain unclear. Although protein phosphorylation clearly affects Shh signaling, little is known about phosphatases governing the pathway. Here, we conducted a small hairpin RNA (shRNA) screen of the phosphatome and identified Eya1 as a positive regulator of Shh signaling. We find that the catalytically active phosphatase Eya1 cooperates with the DNA-binding protein Six1 to promote gene induction in response to Shh and that Eya1/Six1 together regulate Gli transcriptional activators. We show that Eya1, which is mutated in a human deafness disorder, branchio-oto-renal syndrome, is critical for Shh-dependent hindbrain growth and development. Moreover, Eya1 drives the growth of medulloblastoma, a Shh-dependent hindbrain tumor. Together, these results identify Eya1 and Six1 as key components of the Shh transcriptional network in normal development and in oncogenesis.

Eya1 interacts with Six2 and Myc to regulate expansion of the nephron progenitor pool during nephrogenesis.

Self-renewal and proliferation of nephron progenitor cells and the decision to initiate nephrogenesis are crucial events directing kidney development. Despite recent advancements in defining lineage and regulators for the progenitors, fundamental questions about mechanisms driving expansion of the progenitors remain unanswered. Here we show that Eya1 interacts with Six2 and Myc to control self-renewing cell activity. Cell fate tracing reveals a developmental restriction of the Eya1(+) population within the intermediate mesoderm to nephron-forming cell fates and a common origin shared between caudal mesonephric and metanephric nephrons. Conditional inactivation of Eya1 leads to loss of Six2 expression and premature epithelialization of the progenitors. Six2 mediates translocation of Eya1 to the nucleus, where Eya1 uses its threonine phosphatase activity to control Myc phosphorylation/dephosphorylation and function in the progenitor cells. Our results reveal a functional link between Eya1, Six2, and Myc in driving the expansion and maintenance of the multipotent progenitors during nephrogenesis.

Downregulation of CD9 in keratinocyte contributes to cell migration via upregulation of matrix metalloproteinase-9.

Tetraspanin CD9 has been implicated in various cellular and physiological processes, including cell migration. In our previous study, we found that wound repair is delayed in CD9-null mice, suggesting that CD9 is critical for cutaneous wound healing. However, many cell types, including immune cells, endothelial cells, keratinocytes and fibroblasts undergo marked changes in gene expression and phenotype, leading to cell proliferation, migration and differentiation during wound repair, whether CD9 regulates kerationcytes migration directly remains unclear. In this study, we showed that the expression of CD9 was downregulated in migrating keratinocytes during wound repair in vivo and in vitro. Recombinant adenovirus vector for CD9 silencing or overexpressing was constructed and used to infect HaCaT cells. Using cell scratch wound assay and cell migration assay, we have also demonstrated that 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, CD9 silencing-activated JNK signaling was accompanied by the upregulation of MMP-9 activity and expression. Coincidentally, we found that SP600125, a JNK pathway inhibitor, decreased the activity and expression of MMP-9 of CD9-silenced HaCaT cells. Thus, our results suggest that 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.

Phosphorylation of DYNLT1 at serine 82 regulates microtubule stability and mitochondrial permeabilization in hypoxia.

Hypoxia-induced microtubule disruption and mitochondrial permeability transition (mPT) are crucial events leading to fatal cell damage and recent studies showed that microtubules (MTs) are involved in the modulation of mitochondrial function. Dynein light chain Tctex-type 1 (DYNLT1) is thought to be associated with MTs and mitochondria. Previously we demonstrated that DYNLT1 knockdown aggravates hypoxia-induced mitochondrial permeabilization, which indicates a role of DYNLT1 in hypoxic cytoprotection. But the underlying regulatory mechanism of DYNLT1 remains illusive. Here we aimed to investigate the phosphorylation alteration of DYNLT1 at serine 82 (S82) in hypoxia (1% O2). We therefore constructed recombinant adenoviruses to generate S82E and S82A mutants, used to transfect H9c2 and HeLa cell lines. Development of hypoxia-induced mPT (MMP examining, Cyt c release and mPT pore opening assay), hypoxic energy metabolism (cellular viability and ATP quantification), and stability of MTs were examined. Our results showed that phosph-S82 (S82-P) expression was increased in early hypoxia; S82E mutation (phosphomimic) aggravated mitochondrial damage, elevated the free tubulin in cytoplasm and decreased the cellular viability; S82A mutation (dephosphomimic) seemed to diminish the hypoxia-induced injury. These data suggest that DYNLT1 phosphorylation at S82 is involved in MTs and mitochondria regulation, and their interaction and cooperation contribute to the cellular hypoxic tolerance. Thus, we provide new insights into a DYNLT1 mechanism in stabilizing MTs and mitochondria, and propose a potential therapeutic target for hypoxia cytoprotective studies.