Usenamine A induces apoptosis and autophagic cell death of human hepatoma cells via interference with the Myosin-9/actin-dependent cytoskeleton remodeling.

BACKGROUND: Hepatocellular carcinoma (HCC) is a major cause of cancer-associated mortality worldwide. Myosin-9's role in HCC and the anti-HCC effect of the drugs targeting Myosin-9 remain poorly understood so far. Candidate antitumor agents obtained from natural products have attracted worldwide attention. Usenamine A is a novel product, which was first extracted in our laboratory from the lichen Usnea longissima. According to published reports, usenamine A exhibits good antitumor activity, while the mechanisms underlying its antitumor effects remain to be elucidated. PURPOSE: The present study investigated the anti-hepatoma effect of usenamine A and the underlying molecular mechanisms, along with evaluating the therapeutic potential of targeting Myosin-9 in HCC. METHODS: The CCK-8, Hoechst staining, and FACS assays were conducted in the present study to investigate how usenamine A affected the growth and apoptosis of human hepatoma cells. Moreover, TEM, acridine orange staining, and immunofluorescence assay were performed to explore the induction of autophagy by usenamine A in human hepatoma cells. The usenamine A-mediated regulation of protein expression in human hepatoma cells was analyzed using immunoblotting. MS analysis, SPR assay, CETSA, and molecular modeling were performed to identify the direct target of usenamine A. Immunofluorescence assay and co-immunoprecipitation assay were conducted to determine whether usenamine A affected the interaction between Myosin-9 and the actin present in human hepatoma cells. In addition, the anti-hepatoma effect of usenamine A was investigated in vivo using a xenograft tumor model and the IHC analysis. RESULTS: The present study initially revealed that usenamine A could suppress the proliferation of HepG2 and SK-HEP-1 cells (hepatoma cell lines). Furthermore, usenamine A induced cell apoptosis via the activation of caspase-3. In addition, usenamine A enhanced autophagy. Moreover, usenamine A administration could dramatically suppress the carcinogenic ability of HepG2 cells, as evidenced by the nude mouse xenograft tumor model. Importantly, it was initially revealed that Myosin-9 was a direct target of usenamine A. Usenamine A could block cytoskeleton remodeling through the disruption of the interaction between Myosin-9 and actin. Myosin-9 participated in suppressing proliferation while inducing apoptosis and autophagy in response to treatment with usenamine A. In addition, Myosin-9 was revealed as a potential oncogene in HCC. CONCLUSIONS: Usenamine A was initially revealed to suppress human hepatoma cells growth by interfering with the Myosin-9/actin-dependent cytoskeleton remodeling through the direct targeting of Myosin-9. Myosin-9 is, therefore, a promising candidate target for HCC treatment, while usenamine A may be utilized as a possible anti-HCC therapeutic, particularly in the treatment of HCC with aberrant Myosin-9.

Capsaicin receptor TRPV1 maintains quiescence of hepatic stellate cells in the liver via recruitment of SARM1.

BACKGROUND & AIMS: Capsaicin receptor, also known as transient receptor potential vanilloid 1 (TRPV1), is involved in pain physiology and neurogenic inflammation. Herein, we discovered the presence of TRPV1 in hepatic stellate cells (HSCs) and aimed to delineate its function in this cell type and liver fibrosis. METHODS: TRPV1 expression was examined in liver biopsies from patients with liver fibrosis using quantitative real-time PCR and immunostaining. Its contribution to liver fibrosis was examined in Trpv1-/- mice, upon lentiviral delivery of the TRPV1 gene, and in human and mouse primary HSCs, using patch clamp, intracellular Ca2+ mobilization determination, FACS analyses and gain/loss of function experiments. Binding of sterile alpha and Toll/interleukin-1 receptor motif-containing protein 1 (SARM1) to TRPV1 was determined using mass spectrometry, co-immunoprecipitation, surface plasmon resonance, bioluminescence resonance energy transfer, and NanoBiT. RESULTS: TRPV1 mRNA levels are significantly downregulated in patients with liver fibrosis and mouse models, showing a negative correlation with F stage and α-smooth muscle actin expression, a marker of HSC activation. TRPV1 expression and function decrease during HSC activation in fibrotic livers in vivo or during culture. Genetic and pharmacological inhibition of TRPV1 in quiescent HSCs leads to NF-κB activation and pro-inflammatory cytokine production. TRPV1 requires binding of its N-terminal ankyrin repeat domain to the TIR-His583 (Toll/interleukin-1 receptor) domain of SARM1 to prevent HSCs from pro-inflammatory activation. Trpv1-/- mice display increased HSC activation and more severe liver fibrosis, whereas TRPV1 overexpression is antifibrotic in various disease models. CONCLUSION: The antifibrotic properties of TRPV1 are attributed to the prevention of HSC activation via the recruitment of SARM1, which could be an attractive therapeutic strategy against liver fibrosis. IMPACT AND IMPLICATIONS: We identified the neuronal channel protein TRPV1 as a gatekeeper of quiescence in hepatic stellate cells, a key driver of liver fibrogenesis and chronic liver disease. Physiologically expressed in healthy liver and consistently downregulated during liver fibrosis development, its therapeutic re-expression is expected to have few side effects, making it an attractive target diagnostic tool and drug candidate for industry and clinicians.

Network Pharmacology Research and Dual-omic Analyses Reveal the Molecular Mechanism of Natural Product Nodosin Inhibiting Muscle-Invasive Bladder Cancer in Vitro and in Vivo.

Bladder cancer, specifically, muscle-invasive bladder cancer (MIBC), is among the most common malignant tumors. Patients with MIBC who cannot tolerate standard drugs require novel treatments. Targeting apoptosis may help treat cancer, which may be achieved with the use of some natural products. Nodosin, found in Isodon serra (Maxim.) Kudo (known as Xihuangcao), may inhibit bladder cancer cells. Transcriptomics and proteomics dual-omic analyses revealed the network pharmacological mechanism: (1) blocking the S phase by up-regulating RPA2, CLSPN, MDC1, PDCD2L, and E2F6 gene expressions, suppressing cancer cell proliferation; (2) inducing apoptosis and autophagy and restraining ferroptosis by up-regulating HMOX1, G0S2, SQSTM1, FTL, SLC7A11, and AIFM2 gene expressions; (3) preventing cancer cell migration by down-regulating NEXN, LIMA1, CFL2, PALLD, and ITGA3 gene expressions. In vivo, nodosin inhibited bladder cancer cell growth in a model of xenograft tumor in nude mice. This study is the first to report basic research findings on the network pharmacological mechanism of cytotoxicity of bladder cancer cells by nodosin, providing novel evidence for the application of nodosin in the field of oncology; however, other mechanisms may be involved in the effects of nodosin for further research. These findings provide a foundation for the development of novel MIBC drugs.

miR-218-5p in endometrial microenvironment prevents the migration of ectopic endometrial stromal cells by inhibiting LASP1.

BACKGROUND: Our previous two-dimensional electrophoresis experiment showed that the expression of LASP1 in patients with endometriosis was significantly higher than that of control endometrium. However, the molecular mechanism by which LASP1 is regulated in endometriosis/adenomyosis is unknown. METHODS: Herein, qPCR was performed to analyze the expression levels of LASP1 and miR-218-5p between endometriosis (Ems) cells and control cells. Fluorescence in situ hybridization was carried out to measure the expression level of miR-218-5p in ectopic endometrium versus normal endometrium. After miR-218-5p mimic or inhibitor were transfected, the transwell experiment was carried out to see the effect of miR-218-5p on the migration of endometrial stromal cells (ESCs). EdU was used to measure cell proliferation rate. Dual-luciferase reporter assay was used to verify the binding of hsa-miR-218-5p to the 3'UTR of LASP1. Western blot and immunofluorescence analysis were carried out to identify the protein expression pattern of LASP1 and EMT markers in endometrial tissue. RESULTS: The miR-218-5p is mainly secreted from blood vessels and expressed in the muscle layer around the endometrium, which inhibits the expression level of LASP1 by binding the 3'UTR region of LASP1 in normal ESCs. Overexpression of miR-218-5p impedes the epithelial-to-mesenchymal transition (EMT) and prevents the migration of ESCs and the expression of Vimentin in Ems. CONCLUSIONS: Our findings revealed that miR-218-5p in endometrial microenvironment prevents the migration of ectopic endometrial stromal cells by inhibiting LASP1.

Acridinium-conjugated aromatic heterocycles as highly potent FtsZ inhibitors: Design, synthesis, and biological evaluation.

The epidemic of multidrug resistance (MDR) is a serious threat to public health, and new classes of antibiotics with novel mechanisms of action are in critical need. We rationally designed and efficiently synthesized three series of new chemical entities with potential antibacterial activity targeting filamenting temperature-sensitive mutant Z (FtsZ). Evaluation of these compounds against a panel of Gram-positive bacteria including MDR and vancomycin-resistant Enterococcus strains indicated that most compounds showed enhanced antibacterial efficacy, comparable or even superior to the reference drugs. The newly synthesized compounds proved to be substrates of the Escherichia coli efflux pump AcrB, thus affecting the activity. Their structure-activity relationships were summarized in detail. The most potent compound 10f quickly eliminated bacteria in a bactericidal mode, with low susceptibility to induce bacterial resistance. Further mechanistic studies with the BsFtsZ protein revealed that 10f functioned as an effective FtsZ inhibitor through altering the dynamics of FtsZ self-polymerization via a stimulatory mechanism, which leads to inhibition of cell division and cell death. Besides, 10f not only displayed no obvious cytotoxicity to mammalian cells but also had a high efficacy in a murine model of bacteremia in vivo. Regarded as a whole, our findings highlight 10f as a promising new FtsZ-targeting bactericidal agent.

Toxicity assessments and transcriptional effects of monofunctionalized Pt(II) complex under dark and light irradiation condition in Caenorhabditis elegans.

In vivo toxicity of aromatic ring (BODIPY, 1,3,5,7,8-pentamethyl dipyrrin borondifluoride) attached monofunctional Pt(II) complexes mCBP {[cis-Pt(NH3)2Cl] 8-(para-pyridine-methylene),1,3,5,7-tetramethyl dipyrrin borondifluoride}+ Nitrate- and dCBP {[cis-Pt(NH3)2Cl]28-(1,3-pyrimidine-5-methylene),1,3,5,7-tetramethyl dipyrrin borondifluoride}2+ diNitrate2- were tested in Caenorhabditis elegans (C. elegans). dCBP showed promising reactive oxygen ROS (reactive oxygen species) generating capability. This complex resulted reduction of lifespan, body length and egg laying rate under dark and light irradiation in both N2 (wild-type, cisplatin resistant) and ok938 (asna-1, cisplatin sensitive) C. elegans. Expressional change of several key cancer related pathway (JNK (c-Jun N-terminal kinase) and Wnt/ß-catenin (Wingless/Integrated/ß-catenin)) related genes (for instance, jnk-1, wrm-1 and gst-4) were confirmed by RNA sequencing experiments. These transcriptional alternations could explain physiological parameters change in nematode and partially revealed how both Pt(II) based complexes influence cancer related pathways. Furthermore, these associated genes exhibited the function of apoptosis, reduced chemoresistance of cancer cells and most of those expressional changes were linked to extended survival of cancer patients.

Ezrin Peptide Therapy from HIV to COVID: Inhibition of Inflammation and Amplification of Adaptive Anti-Viral Immunity.

Human Ezrin Peptides (HEPs) are inhibitors of expression of IL-6 and other inflammatory cytokines, amplifiers of adaptive B cell and T cell immunity and enhancers of tissue repair. The mutation stable C-terminus of HIV gp120, mimics 69% of the "Hep-receptor", a zipped α-helical structure in the middle of the α domain of human ezrin protein. Synthetic peptides homologous to the Hep-receptor of ezrin of five to fourteen amino acids, activate anti-viral immunity against a wide range of viruses (HIV, HCV, herpes, HPV, influenza and other human respiratory viruses). Human Ezrin Peptide One (HEP1) TEKKRRETVEREKE (brand name Gepon, registered for human use in Russia from 2001) is a successful treatment for opportunistic infections in HIV-infected patients. That treats HEP1and prevents mucosal candidiasis, herpes zoster outbreaks and infection-induced chronic diarrhea. There are clinical publications in Russian on the successful treatments of chronic recurrent vaginal candidiasis, acute and chronic enterocolitis and dysbacteriosis, which are accompanied by normalization of the mucosal microbiome, and the decline or disappearance of inflammation. HEP1 is also an effective treatment and prevention for recurrent inflammation and ulceration in the stomach, duodenum and colon. HEP1 and RepG3 GEKKRRETVEREGG (a derivative of HEP1) have been used successfully as an inhaled spray peptide solution to treat a small number of human volunteers with mild-to-moderate COVID, resulting from SARS-CoV-2 infection, based on earlier successes in treating acute viral respiratory disease with inflammatory complications. Ezrin peptides seem to correct a dysregulation of innate immune responses to SARS-CoV-2. They are also adjuvants of B cell adaptive immunity and increase antibody titres, resulting in protection from lethal virus infection of mice. In a clinical study in Moscow, orally administered HEP1 was shown to enhance antibody-titres produced in response to hepatitis-B vaccination. These very preliminary but promising results with ezrin peptide treatment of COVID must be replicated in large-scale randomised placebo controlled clinical studies, to be verified.

Proline-Serine-Threonine Phosphatase-Interacting Protein 2 Alleviates Diabetes Mellitus-Osteoarthritis in Rats through Attenuating Synovial Inflammation and Cartilage Injury.

OBJECTIVE: To explore the possible way of proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2) influencing diabetes mellitus-osteoarthritis (DM-OA) progression. METHODS: In vivo, eight-week-old male Sprague Dawley rats were induced with DM-OA by intraperitoneal injection of streptozotocin with high-fat diet feeding and intra-articular injection of monoiodoacetate. PSTPIP2 overexpression was achieved by intra-articular injection of lentivirus vectors. PSTPIP2 expression was verified by real-time polymerase chain reaction and Western blotting. Histological changes were examined by hematoxylin/eosin and safranin-O/fast-green staining. In vitro, rat synovial fibroblasts were induced DM-OA by stimulation of high glucose (HG) and interleukin (IL)-1ß. PSTPIP2 overexpression was achieved by lentivirus infection. U0126 was added as an ERK inhibitor. Levels of tumor necrosis factor (TNF)-α, IL-6, and IL-1ß were detected using enzyme-linked immunosorbent assay. Expression of matrix metalloproteinase (MMP)-3, MMP-13, aggrecanase-2 (ADAMTS-5), intercellular cell adhesion molecule (ICAM)-1, extracellular regulated protein kinase (ERK) and phospho-ERK (p-ERK) was detected by Western blotting. RESULTS: In DM-OA rats, PSTPIP2 relative messenger RNA (mRNA) level was significantly decreased compared to control rats. The protein expression was also decreased obviously. Inflammation score in synovium was dramatically increased, accompanying with increased TNF-α, IL-6, and IL-1ß levels. Osteoarthritis research society international (OARSI) score in cartilage was markedly increased, along with increased MMP-3, MMP-13, ADAMTS-5, ICAM-1, ERK and p-ERK expression. In PSTPIP2-overexpressed DM-OA rats, PSTPIP2 mRNA level and protein expression was increased compared to DM-OA rats received negative-control lentivirus vectors. The inflammation score, as well as TNF-α, IL-6, and IL-1ß levels were dramatically decreased. Also, the OARSI score and protein expression of MMP-3, MMP-13, ADAMTS-5, ICAM-1, ERK and p-ERK were decreased. In HG+IL-1ß-treated rat synovial fibroblasts, PSTPIP2 protein expression was decreased compared to normal glucose (NG)-treated cells. Levels of TNF-α, IL-6, and IL-1ß, as well as expression of MMP-3, MMP-13, ADAMTS-5, ICAM-1, ERK and p-ERK were increased. After cells were infected with PSTPIP2-overexpressed lentivirus, levels of TNF-α, IL-6, and IL-1ß, and expression of MMP-3, MMP-13, ADAMTS-5, ICAM-1, ERK and p-ERK were obviously decreased compared to cells infected with NC lentivirus. In addition, ERK inhibitor U0126 treatment also decreased the TNF-α, IL-6, and IL-1ßlevels and MMP-3, MMP-13, ADAMTS-5, ICAM-1, ERK and p-ERK expression in HG + IL-1ß treated rat synovial fibroblasts. CONCLUSION: Overexpression of PSTPIP2 alleviates synovial inflammation and cartilage injury during DM-OA progression via inhibiting ERK phosphorylation.

Ezrin protects bovine spermatozoa from spontaneous acrosome reaction.

To interact and penetrate the egg, the spermatozoon must undergo a maturation step called the acrosome reaction (AR) in close proximity to the egg. This process can take place only after a series of biochemical changes to the sperm occur in the female reproductive tract, collectively called capacitation. Spermatozoa can undergo spontaneous-acrosome reaction (sAR) before reaching the vicinity of the egg, preventing successful fertilization. Several mechanisms were shown to protect spermatozoa from undergoing sAR. Here we describe the involvement of the actin cross-linker, Ezrin in the mechanism that protects spermatozoa from sAR. Inhibition of Ezrin stimulates sAR and inhibits actin polymerization. Ezrin is highly phosphorylated/activated during the first hour of the capacitation process, and its phosphorylation rate is subsequently decreased. Ezrin phosphorylation depends on protein kinase A (PKA) and calmodulin kinase II (CaMKII) activities, and to some extent on phosphatidyl-inositol-4-kinase (PI4K) activity. Inhibition of these three kinases stimulates sAR, in which the effect of PI4K inhibition, but not PKA or CaMKII inhibition, can be reversed by increasing p-Ezrin using a phosphatase inhibitor. All together, we showed that three kinases mediate Ezrin activation during spermatozoa capacitation, leading to actin polymerization in a mechanism that prevents sAR.

Therapeutic Effects of Hyaluronic Acid in Peritonitis-Induced Sepsis in Mice.

Intra-abdominal infection is the second most common cause of sepsis, and the mortality rate from abdominal sepsis remains high. High molecular weight (HMW) hyaluronic acid (HA) has been studied in sterile injury models as an anti-inflammatory and anti-permeability agent. This study evaluated the therapeutic effects of intraperitoneal HMW HA administration in mice with peritonitis-induced sepsis. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP), followed 4 h later by an intraperitoneal injection of HMW HA (20 mg/kg) solution or phosphate buffered saline (PBS). Survival, physiological data, organ injury, bacterial burden, and inflammatory cytokine levels were assessed in the CLP mice. To assess the effect of HA on macrophage phagocytosis activity, RAW264.7 cells, primed with lipopolysaccharide, were exposed with either PBS or HMW HA (500 µg/mL) prior to exposure to 10 CFU of E coli bacteria. HMW HA instillation significantly improved blood oxygenation, lung histology, and survival in CLP mice. Inflammatory cytokine levels in the plasma and bacterial burdens in the lung and spleen were significantly decreased by HA administration at 24 h after CLP. At 6 h after CLP, HA significantly decreased bacterial burden in the peritoneal lavage fluid. HMW HA administration significantly increased E coli bacterial phagocytosis by RAW264.7 cells in part through increased phosphorylation of ezrin/radixin/moesin, a known downstream target of CD44 (a HA receptor); ezrin inhibition abolished the enhanced phagocytosis by RAW264.7 cells induced by HA. Intraperitoneal administration of HMW HA had therapeutic effects against CLP-induced sepsis in terms of suppressing inflammation and increasing antimicrobial activity.

Discovery of 1,3,4-oxadiazol-2-one-containing benzamide derivatives targeting FtsZ as highly potent agents of killing a variety of MDR bacteria strains.

The spread of infections caused by multidrug-resistant (MDR) pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA), has created a need for new antibiotics with novel mechanisms of action. The bacterial division protein FtsZ has been identified as a novel drug target that can be exploited clinically. As part of an ongoing effort to develop FtsZ-targeting antibacterial agents, we describe herein the design, synthesis and bioactivity of six series of novel 1,3,4-oxadiazol-2-one-containing, 1,2,4-triazol-3-one-containing and pyrazolin-5-one-containing benzamide derivatives. Among them, compound A14 was found to be the most potent antibacterial agent, much better than clinical drugs such as ciprofloxacin, linezolid and erythromycin against all the tested gram-positive strains, particularly methicillin-resistant, penicillin-resistant and clinical isolated S. aureus. Subsequent studies on biological activities and docking analyses proved that A14 functioned as an effective compound targeting FtsZ. Preliminary SAR indicated a general direction for further optimization of these novel analogues. Taken together, this research provides a promising chemotype for developing newer FtsZ-targeting bactericidal agents.

An ARC-Regulated IL1ß/Cox-2/PGE2/ß-Catenin/ARC Circuit Controls Leukemia-Microenvironment Interactions and Confers Drug Resistance in AML.

The apoptosis repressor with caspase recruitment domain (ARC) protein is a strong independent adverse prognostic marker in acute myeloid leukemia (AML). We previously reported that ARC regulates leukemia-microenvironment interactions through the NFκB/IL1ß signaling network. Malignant cells have been reported to release IL1ß, which induces PGE2 synthesis in mesenchymal stromal cells (MSC), in turn activating ß-catenin signaling and inducing the cancer stem cell phenotype. Although Cox-2 and its enzymatic product PGE2 play major roles in inflammation and cancer, the regulation and role of PGE2 in AML are largely unknown. Here, we report that AML-MSC cocultures greatly increase Cox-2 expression in MSC and PGE2 production in an ARC/IL1ß-dependent manner. PGE2 induced the expression of ß-catenin, which regulated ARC and augmented chemoresistance in AML cells; inhibition of ß-catenin decreased ARC and sensitized AML cells to chemotherapy. NOD/SCIDIL2RγNull-3/GM/SF mice transplanted with ARC-knockdown AML cells had significantly lower leukemia burden, lower serum levels of IL1ß/PGE2, and lower tissue human ARC and ß-catenin levels, prolonged survival, and increased sensitivity to chemotherapy than controls. Collectively, we present a new mechanism of action of antiapoptotic ARC by which ARC regulates PGE2 production in the tumor microenvironment and microenvironment-mediated chemoresistance in AML.Significance: The antiapoptotic protein ARC promotes AML aggressiveness by enabling detrimental cross-talk with bone marrow mesenchymal stromal cells.

Urokinase-type plasminogen activator (uPA) protects the tripartite synapse in the ischemic brain via ezrin-mediated formation of peripheral astrocytic processes.

Cerebral ischemia has a harmful effect on the synapse associated with neurological impairment. The "tripartite synapse" is assembled by the pre- and postsynaptic terminals, embraced by astrocytic elongations known as peripheral astrocytic processes (PAPs). Ischemic stroke induces the detachment of PAPs from the synapse, leading to synaptic dysfunction and neuronal death. Ezrin is a membrane-associated protein, required for the formation of PAPs, that links the cell surface to the actin cytoskeleton. Urokinase-type plasminogen activator (uPA) is a serine proteinase that upon binding to its receptor (uPAR) promotes neurite growth during development. In the adult brain, neurons release uPA and astrocytes recruit uPAR to the plasma membrane during the recovery phase from an ischemic stroke, and uPA/uPAR binding promotes functional improvement following an ischemic injury. We found that uPA induces the synthesis of ezrin in astrocytes, with the subsequent formation of PAPs that enter in direct contact with the synapse. Furthermore, either the release of neuronal uPA or intravenous treatment with recombinant uPA (ruPA) induces the formation of PAPs in the ischemic brain, and the interaction of these PAPs with the pre- and postsynaptic terminals protects the integrity of the "tripartite synapse" from the harmful effects of the ischemic injury.

Hydrogen peroxide-inducible clone-5 regulates mesangial cell proliferation in proliferative glomerulonephritis in mice.

Hydrogen peroxide-inducible clone-5 (Hic-5) is a transforming growth factor (TGF)-ß1-inducible focal adhesion protein. We previously demonstrated that Hic-5 was localized in mesangial cells and its expression was associated with glomerular cell proliferation and matrix expansion in human and rat glomerulonephritis (GN). In the present study, we first assessed the role of Hic-5 in mesangioproliferative GN by injecting Habu venom into heminephrectomized wild type (Hic-5+/+) and Hic-5-deficient (Hic-5-/-) mice. Hic-5+/+ GN mice exhibited glomerular cell proliferation on day 7. Surprisingly, glomerular cell number and Ki-67-positive cells in Hic-5-/- GN mice were significantly greater than those in Hic-5+/+ GN mice on day 7, although the number of glomerular apoptotic cells and the expression of growth factors (platelet-derived growth factor-BB and TGF-ß1) and their receptors were similarly increased in both Hic-5+/+ and Hic-5-/- GN mice. In culture experiments, proliferation assays showed that platelet-derived growth factor-BB and TGF-ß1 enhanced the proliferation of Hic-5-/- mesangial cells compared with Hic-5+/+ mesangial cells. In addition, mitogenic regulation by Hic-5 was associated with altered and coordinated expression of cell cycle-related proteins including cyclin D1 and p21. The present results suggest that Hic-5 might regulate mesangial cell proliferation in proliferative GN in mice. In conclusion, modulation of Hic-5 expression might have a potential to prevent mesangial cell proliferation in the acute mitogenic phase of glomerulonephritis.

Thermally labile components of aqueous humor potently induce osteogenic potential in adipose-derived mesenchymal stem cells.

Adipose-derived mesenchymal stem cells (ASCs) hold promise for use in cell-based therapies. Their intrinsic anti-inflammatory properties are potentially useful for treatments of inflammatory conditions such as uveitis, while their ability to differentiate along multiple cell lineages suggests use in regenerating damaged or degenerated tissue. However, how ASCs will respond to the intraocular environment is poorly studied. We have recently reported that aqueous humor (AH), the fluid that nourishes the anterior segment of the eye, potently increases alkaline phosphatase (ALP) activity of ASCs, indicating osteogenic differentiation. Here, we expand on our previous findings to better define the nature of this response. To this end, we cultured ASCs in the presence of 0, 5, 10, and 20% AH and assayed them for ALP activity. We found ALP activity correlates with increasing AH concentrations from 5 to 20%, and that longer treatments result in increased ALP activity. By using serum free media and pretreating AH with dextran-coated charcoal, we found that serum and charcoal-adsorbable AH components augment but are not required for this response. Further, by heat-treating the AH, we established that thermally labile components are required for the osteogenic response. Finally, we showed myocilin, a protein present in AH, could induce ALP activity in ASCs. However, this was to a lesser extent than untreated 5% AH, and myocilin could only partially rescue the effect after heat treatment, documenting there were additional thermally labile constituents of AH involved in the osteogenic response. Our work adds to the understanding of the induction of ALP in ASCs following exposure to AH, providing important insight in how ASCs will be influenced by the ocular environment. In conclusion, increased osteogenic potential upon exposure to AH represents a potential challenge to developing ASC cell-based therapies directed at the eye.

Cancer-testis antigen HCA587/MAGE-C2 interacts with BS69 and promotes its degradation in the ubiquitin-proteasome pathway.

HCA587, also known as MAGE-C2, belonging to the MAGE gene family which is characterized by a conserved MAGE Homology Domain, is active in various types of tumors and silent in normal tissues except in male germ-line cells. The biological function of HCA587 is largely unknown. To analyze it, we attempted to identify protein partners of HCA587. We immunopurified HCA587-containing complex from HEK293 cells and identified BS69, a potential tumor suppressor, as an associated protein by mass spectrometry, and the following Immunoprecipitation and GST pull-down assays confirmed HCA587 interaction with BS69. Interestingly, overexpression of HCA587 promoted ubiquitination and the proteasomal degradation of BS69 whereas knockdown of endogenous HCA587 increased the protein level of BS69. Consistent with a functional role for BS69 in negatively regulating LMP1-induced NF-κB activation, overexpression of HCA587 resulted in a significant enhancement of LMP1-induced IL-6 production. These data indicate that HCA587 is a new negative regulator of BS69.

Attenuation of flightless I improves wound healing and enhances angiogenesis in a murine model of type 1 diabetes.

AIMS/HYPOTHESIS: Skin lesions and ulcerations are severe complications of diabetes that often result in leg amputations. In this study we investigated the function of the cytoskeletal protein flightless I (FLII) in diabetic wound healing. We hypothesised that overexpression of FLII would have a negative effect on diabetic wound closure and modulation of this protein using specific FLII-neutralising antibodies (FnAb) would enhance cellular proliferation, migration and angiogenesis within the diabetic wound. METHODS: Using a streptozotocin-induced model of diabetes we investigated the effect of altered FLII levels through Flii genetic knockdown, overexpression or treatment with FnAb on wound healing. Diabetic wounds were assessed using histology, immunohistochemistry and biochemical analysis. In vitro and in vivo assays of angiogenesis were used to assess the angiogenic response. RESULTS: FLII levels were elevated in the wounds of both diabetic mice and humans. Reduction in the level of FLII improved healing of murine diabetic wounds and promoted a robust pro-angiogenic response with significantly elevated von Willebrand factor (vWF) and vascular endothelial growth factor (VEGF)-positive endothelial cell infiltration. Diabetic mouse wounds treated intradermally with FnAb showed improved healing and a significantly increased rate of re-epithelialisation. FnAb improved the angiogenic response through enhanced formation of capillary tubes and functional neovasculature. Reducing the level of FLII led to increased numbers of mature blood vessels, increased recruitment of smooth muscle actin-α-positive cells and improved tight junction formation. CONCLUSIONS/INTERPRETATION: Reducing the level of FLII in a wound may be a potential therapeutic approach for the treatment of diabetic foot ulcers.

Activation of the heat shock response attenuates the interleukin 1ß-mediated inhibition of the amiloride-sensitive alveolar epithelial ion transport.

Acute lung injury (ALI) is a clinical syndrome characterized by hypoxia, which is caused by the breakdown of the alveolar capillary barrier. Interleukin 1ß (IL-1ß), a cytokine released within the airspace in ALI, downregulates the α subunit of the epithelial sodium channel (αENaC) transcription and protein expression via p38 MAP kinase-dependent signaling. Although induction of the heat shock response can restore alveolar fluid clearance compromised by IL-1ß following the onset of severe hemorrhagic shock in rats, the mechanisms are not fully understood. In this study, we report that the induction of the heat shock response prevents IL-1ß-dependent inhibition of αENaC mRNA expression and subsequent channel function. Heat shock results in IRAK1 detergent insolubility and a disruption of Hsp90 binding to IRAK1. Likewise, TAK1, another client protein of Hsp90 and signaling component of the IL-1ß pathway, is also detergent insoluble after heat shock. Twenty-four hours after heat shock, both IRAK1 and TAK1 are again detergent soluble, which correlates with the IL-1ß-dependent p38 activation. Remarkably, IL-1ß-dependent p38 activation 24 h after heat shock did not result in an inhibition of αENaC mRNA expression and channel function. Further analysis demonstrates prolonged preservation of αENaC expression by the activation of the heat shock response that involves inducible Hsp70. Inhibition of Hsp70 at 24 h after heat shock results in p38-dependent IL-1ß inhibition of αENaC mRNA expression, whereas overexpression of Hsp70 attenuates the p38-dependent IL-1ß inhibition of αENaC mRNA expression. These studies demonstrate new mechanisms by which the induction of the heat shock response protects the barrier function of the alveolar epithelium in ALI.