α-Smooth muscle actin is an inconsistent marker of fibroblasts responsible for force-dependent TGFß activation or collagen production across multiple models of organ fibrosis.

Fibrosis is a common pathological sequela of tissue injury or inflammation, and is a major cause of organ failure. Subsets of fibroblasts contribute to tissue fibrosis in multiple ways, including generating contractile force to activate integrin-bound, latent TGFß and secreting excess amounts of collagens and other extracellular matrix proteins (ECM) that make up pathologic scar. However, the precise fibroblast subsets that drive fibrosis have been poorly understood. In the absence of well-characterized markers, α-smooth muscle actin (αSMA) is often used to identify pathologic fibroblasts, and some authors have equated αSMA(+) cells with contractile myofibroblasts and proposed that these cells are the major source of ECM. Here, we investigated how well αSMA expression describes fibroblast subsets responsible for TGFß activation and collagen production in three commonly used models of organ fibrosis that we previously reported could be inhibited by loss of αv integrins on all fibroblasts (using PDGFRß-Cre). Interestingly, αSMA-directed deletion of αv integrins protected mice from CCl4-induced hepatic fibrosis, but not bleomycin-induced pulmonary or unilateral ureteral obstruction-induced renal fibrosis. Using Col-EGFP/αSMA-RFP dual reporter mice, we found that only a minority of collagen-producing cells coexpress αSMA in the fibrotic lung and kidney. Notably, Col-EGFP(+)αSMA-RFP(-) cells isolated from the fibrotic lung and kidney were equally capable of activating TGFß as were Col-EGFP(+)αSMA-RFP(+) cells from the same organ, and this TGFß activation was blocked by a TGFß-blocking antibody and an inhibitor of nonmuscle myosin, respectively. Taken together, our results suggest that αSMA is an inconsistent marker of contractile and collagen-producing fibroblasts in murine experimental models of organ fibrosis.

Four New Tirucallane Triterpenoids from the Fruits of Melia azedarach and Their Cytotoxic Activities.

Four new tirucallane triterpenoids, (21S,23R,24R)-21,23-epoxy-21,24-dihydroxy-25-methoxytirucall-7-en-3-one (2), (3S,21S,23R,24S)-21,23-epoxy-21,25-dimethoxytirucall-7-ene-3,24-diol (8), (21S,23R,24R)-21,23-epoxy-24-hydroxy-21-methoxytirucalla-7,25-dien-3-one (11), and (21S,23R,24R)-21,23-epoxy-21,24-dihydroxytirucalla-7,25-dien-3-one (12), along with 16 known analogues, 1, 3 - 7, 9 - 10, and 13 - 20, were isolated from the fruits of Melia azedarach. Their structures were elucidated by spectroscopic methods including 1D- and 2D-NMR techniques and mass spectrometry. These compounds were evaluated for their cytotoxicities against HepG2 (liver), SGC7901 (stomach), K562 (leukemia), and HL60 (leukemia) cancer cell lines. Compound 20 exhibited potent cytotoxicity against HepG2 and SGC7901 cancer cells with the IC50 values of 6.9 and 6.9 µm, respectively.

Glutathione-S-transferase P1 is a critical regulator of Cdk5 kinase activity.

Cyclin dependent kinase-5 (Cdk5) activity is deregulated in Alzheimer's disease (AD) and contributes to all three hallmarks: neurotoxic ß-amyloid formation, neurofibrillary tangles, and neuronal death. However, the mechanism leading to Cdk5 deregulation remains controversial. Cdk5 deregulation in AD is usually linked to the formation of p25, a proteolysis product of Cdk5 activator p35, which leads to Cdk5 mislocalization and hyperactivation. A few studies have indeed shown increased p25 levels in AD brains; however, others have refuted this observation. These contradictory findings suggest that additional factors contribute to Cdk5 deregulation. This study identified glutathione-S-transferase pi 1 (GSTP1) as a novel Cdk5 regulatory protein. We demonstrate that it is a critical determinant of Cdk5 activity in human AD brains and various cancer and neuronal cells. Increased GSTP1 levels were consistently associated with reduced Cdk5 activity. GSTP1 directly inhibits Cdk5 by dislodging p25/p35, and indirectly by eliminating oxidative stress. Cdk5 promotes and is activated by oxidative stress, thereby engaging a feedback loop which ultimately leads to cell death. Not surprisingly, GSTP1 transduction conferred a high degree of neuroprotection under neurotoxic conditions. Given the critical role of oxidative stress in AD pathogenesis, an increase in GSTP1 level may be an alternative way to modulate Cdk5 signaling, eliminate oxidative stress, and prevent neurodegeneration.

Molecular mapping of interstitial lung disease reveals a phenotypically distinct senescent basal epithelial cell population.

Compromised regenerative capacity of lung epithelial cells can lead to cellular senescence, which may precipitate fibrosis. While increased markers of senescence have been reported in idiopathic pulmonary fibrosis (IPF), the origin and identity of these senescent cells remain unclear, and tools to characterize context-specific cellular senescence in human lung are lacking. We observed that the senescent marker p16 is predominantly localized to bronchiolized epithelial structures in scarred regions of IPF and systemic sclerosis-associated interstitial lung disease (SSc-ILD) lung tissue, overlapping with the basal epithelial markers Keratin 5 and Keratin 17. Using in vitro models, we derived transcriptional signatures of senescence programming specific to different types of lung epithelial cells and interrogated these signatures in a single-cell RNA-Seq data set derived from control, IPF, and SSc-ILD lung tissue. We identified a population of basal epithelial cells defined by, and enriched for, markers of cellular senescence and identified candidate markers specific to senescent basal epithelial cells in ILD that can enable future functional studies. Notably, gene expression of these cells significantly overlaps with terminally differentiating cells in stratified epithelia, where it is driven by p53 activation as part of the senescence program.

Embryonic lethality and defective mammary gland development of activator-function impaired conditional knock-in Erbb3 V943R mice.

ERBB3 is a pseudokinase domain-containing member of the ERBB family of receptor tyrosine kinases (RTKs). Following ligand binding, ERBB receptors homo- or hetero-dimerize, leading to a head-to-tail arrangement of the intracellular kinase domains, where the "receiver" kinase domain of one ERBB is activated by the "activator" domain of the other ERBB in the dimer. In ERBB3, a conserved valine at codon 943 (V943) in the kinase C-terminal domain has been shown to be important for its function as an "activator" kinase in vitro. Here we report a knock-in mouse model where we have modified the endogenous Erbb3 allele to allow for tissue-specific conditional expression of Erbb3 V943R (Erbb3 CKI-V943R ). Additionally, we generated an Erbb3 D850N (Erbb3 CKI-D850N ) conditional knock-in mouse model where the conserved aspartate in the DFG motif of the pseudokinase domain was mutated to abolish any potential residual kinase activity. While Erbb3 D850N/D850N animals developed normally, homozygous Erbb3 V943R/V943R expression during development resulted in embryonic lethality. Further, tissue specific expression of Erbb3 V943R/V943R in the mammary gland epithelium following its activation using MMTV-Cre resulted in delayed elongation of the ductal network during puberty. Single-cell RNA-seq analysis of Erbb3 V943R/V943R mammary glands showed a reduction in a specific subset of fibrinogen-producing luminal epithelial cells.

Dual targeting of lymphocyte homing and retention through α4ß7 and αEß7 inhibition in inflammatory bowel disease.

Anti-integrins are therapeutically effective for inflammatory bowel disease, yet the relative contribution of α4ß7 and αEß7 to gut lymphocyte trafficking is not fully elucidated. Here, we evaluate the effect of α4ß7 and αEß7 blockade using a combination of murine models of gut trafficking and longitudinal gene expression analysis in etrolizumab-treated patients with Crohn's disease (CD). Dual blockade of α4ß7 and αEß7 reduces CD8+ T cell accumulation in the gut to a greater extent than blockade of either integrin alone. Anti-αEß7 reduces epithelial:T cell interactions and promotes egress of activated T cells from the mucosa into lymphatics. Inflammatory gene expression is greater in human intestinal αEß7+ T cells. Etrolizumab-treated patients with CD display a treatment-specific reduction in inflammatory and cytotoxic intraepithelial lymphocytes (IEL) genes. Concurrent blockade of α4ß7 and αEß7 promotes reduction of cytotoxic IELs and inflammatory T cells in the gut mucosa through a stepwise inhibition of intestinal tissue entry and retention.

TGFß2 and TGFß3 isoforms drive fibrotic disease pathogenesis.

Transforming growth factor-ß (TGFß) is a key driver of fibrogenesis. Three TGFß isoforms (TGFß1, TGFß2, and TGFß3) in mammals have distinct functions in embryonic development; however, the postnatal pathological roles and activation mechanisms of TGFß2 and TGFß3 have not been well characterized. Here, we show that the latent forms of TGFß2 and TGFß3 can be activated by integrin-independent mechanisms and have lower activation thresholds compared to TGFß1. Unlike TGFB1, TGFB2 and TGFB3 expression is increased in human lung and liver fibrotic tissues compared to healthy control tissues. Thus, TGFß2 and TGFß3 may play a pathological role in fibrosis. Inducible conditional knockout mice and anti-TGFß isoform-selective antibodies demonstrated that TGFß2 and TGFß3 are independently involved in mouse fibrosis models in vivo, and selective TGFß2 and TGFß3 inhibition does not lead to the increased inflammation observed with pan-TGFß isoform inhibition. A cocrystal structure of a TGFß2-anti-TGFß2/3 antibody complex reveals an allosteric isoform-selective inhibitory mechanism. Therefore, inhibiting TGFß2 and/or TGFß3 while sparing TGFß1 may alleviate fibrosis without toxicity concerns associated with pan-TGFß blockade.

Integrin αvß8 on T cells suppresses anti-tumor immunity in multiple models and is a promising target for tumor immunotherapy.

αvß8 integrin, a key activator of transforming growth factor ß (TGF-ß), inhibits anti-tumor immunity. We show that a potent blocking monoclonal antibody against αvß8 (ADWA-11) causes growth suppression or complete regression in syngeneic models of squamous cell carcinoma, mammary cancer, colon cancer, and prostate cancer, especially when combined with other immunomodulators or radiotherapy. αvß8 is expressed at the highest levels in CD4+CD25+ T cells in tumors, and specific deletion of ß8 from T cells is as effective as ADWA-11 in suppressing tumor growth. ADWA-11 increases expression of a suite of genes in tumor-infiltrating CD8+ T cells normally inhibited by TGF-ß and involved in tumor cell killing, including granzyme B and interferon-γ. The in vitro cytotoxic effect of tumor CD8 T cells is inhibited by CD4+CD25+ cells, and this suppressive effect is blocked by ADWA-11. These findings solidify αvß8 integrin as a promising target for cancer immunotherapy.

Characteristics delineation of piscidin 5 like from Larimichthys crocea with evidence for the potent antiparasitic activity.

Several researches reported that piscidin members of teleosts owned strong antiparasitic activity. Cryptocaryon irritans, a type of ectoparasite, could infect most of the marine teleosts. Larimichthys crocea could severely suffer from marine white spot disease caused by C. irritans, and their mortality rate was significantly high. Concentrating on this problem, we have done many related works. Piscidin 5 like (termed Lc-P5L) was another piscidin member isolated from a comparative transcriptome of C. irritans-immuned L. crocea. In the paper, quantitative Real-time PCR (qRT-PCR) showed Lc-P5L was upregulated in examined tissues, including gill, head kidney, muscle, liver, spleen and intestine after challenged by C. irritans, the significant upregulation time was in accordance to key developmental stages of C. irritans, which implied different infection stages could result in host immune response. Furthermore, using microscope techniques, we observed theronts or trophonts became weakly motile, cilia became detached, cells were out of shape, membranes eventually lysed in different cell positions and cytoplasmic contents leaked. Laser confocal scanning microscope (LCSM) observed theronts macronucleus grew swell and depolymerized after treated by recombinant Lc-P5L (rLc-P5L). Data suggested rLc-P5L was significantly lethal to C. irritans, and the death state of the parasite incubated with rLc-P5L was remarkably similar to other piscidin members or other antiparasitic peptides (APPs). Thus, these data provided new insights into L. crocea immunity against C. irritans and potential of rLc-P5L as a therapeutic agent against pathogen invasion.

Osteopontin Links Myeloid Activation and Disease Progression in Systemic Sclerosis.

Progressive lung fibrosis is a major cause of mortality in systemic sclerosis (SSc) patients, but the underlying mechanisms remain unclear. We demonstrate that immune complexes (ICs) activate human monocytes to promote lung fibroblast migration partly via osteopontin (OPN) secretion, which is amplified by autocrine monocyte colony stimulating factor (MCSF) and interleukin-6 (IL-6) activity. Bulk and single-cell RNA sequencing demonstrate that elevated OPN expression in SSc lung tissue is enriched in macrophages, partially overlapping with CCL18 expression. Serum OPN is elevated in SSc patients with interstitial lung disease (ILD) and prognosticates future lung function deterioration in SSc cohorts. Serum OPN levels decrease following tocilizumab (monoclonal anti-IL-6 receptor) treatment, confirming the connection between IL-6 and OPN in SSc patients. Collectively, these data suggest a plausible link between autoantibodies and lung fibrosis progression, where circulating OPN serves as a systemic proxy for IC-driven profibrotic macrophage activity, highlighting its potential as a promising biomarker in SSc ILD.

Collagen-producing lung cell atlas identifies multiple subsets with distinct localization and relevance to fibrosis.

Collagen-producing cells maintain the complex architecture of the lung and drive pathologic scarring in pulmonary fibrosis. Here we perform single-cell RNA-sequencing to identify all collagen-producing cells in normal and fibrotic lungs. We characterize multiple collagen-producing subpopulations with distinct anatomical localizations in different compartments of murine lungs. One subpopulation, characterized by expression of Cthrc1 (collagen triple helix repeat containing 1), emerges in fibrotic lungs and expresses the highest levels of collagens. Single-cell RNA-sequencing of human lungs, including those from idiopathic pulmonary fibrosis and scleroderma patients, demonstrate similar heterogeneity and CTHRC1-expressing fibroblasts present uniquely in fibrotic lungs. Immunostaining and in situ hybridization show that these cells are concentrated within fibroblastic foci. We purify collagen-producing subpopulations and find disease-relevant phenotypes of Cthrc1-expressing fibroblasts in in vitro and adoptive transfer experiments. Our atlas of collagen-producing cells provides a roadmap for studying the roles of these unique populations in homeostasis and pathologic fibrosis.

Two new phenylspirodrimanes from the deep-sea derived fungus Stachybotrys sp. MCCC 3A00409.

Two new phenylspirodrimanes, stachybotrin H (1) and stachybotrysin H (9) together with eleven known analogues (2-8, 10-13) were isolated from deep-sea derived Stachybotrys sp. MCCC 3A00409. Their structures were determined by extensive NMR data and mass spectroscopic analysis. Compounds 9-12 showed weak cytotoxicity against three human cancer cell lines K562, Hela and HL60 with IC50 in the range of 18.5-52.8 µM.

A new indole-type alkaloid from the roots of Clematis florida var. plena.

One new indole-type alkaloid, α-L-rhamnopyranosyl-(1→6)-ß-D- glucopyranosyl 6-methoxy-3-indolecarbonate (1), together with three known alkaloids (2-4), one aromatic acid (5) and five known saponins (6-10), was isolated from the roots of Clematis florida var. plena. Their structures were established by NMR spectroscopic analysis and acid hydrolysis. In in vivo anti-inflammatory activity, n-butanol extract was found to be potent against ear edema in mice, with inhibition rate of 48.7% at a dose of 800 mg/kg. Furthermore, compounds 8 and 9 obtained from the n-butanol extract exhibited significant anti-inflammatory activities with inhibition rates of 50.9% and 54.7% at a dose of 200 mg/kg.

Deregulated Cdk5 promotes oxidative stress and mitochondrial dysfunction.

Oxidative stress is one of the earliest events in Alzheimer's disease (AD). A chemical genetic screen revealed that deregulated cyclin-dependent kinase 5 (Cdk5) may cause oxidative stress by compromising the cellular anti-oxidant defense system. Using novel Cdk5 modulators, we show the mechanism by which Cdk5 can induce oxidative stress in the disease's early stage and cell death in the late stage. Cdk5 dysregulation upon neurotoxic insults results in reactive oxygen species (ROS) accumulation in neuronal cells because of the inactivation of peroxiredoxin I and II. Sole temporal activation of Cdk5 also increases ROS, suggesting its major role in this process. Cdk5 inhibition rescues mitochondrial damage upon neurotoxic insults, thereby revealing Cdk5 as an upstream regulator of mitochondrial dysfunction. As mitochondrial damage results in elevated ROS and Ca(2+) levels, both of which activate Cdk5, we propose that a feedback loop occurs in late stage of AD and leads to cell death (active Cdk5 --> ROS --> excess ROS --> mitochondrial damage --> ROS --> hyperactive Cdk5 --> severe oxidative stress and cell injury --> cell death). Cdk5 inhibition upon neurotoxic insult prevents cell death significantly, supporting this hypothesis. As oxidative stress and mitochondrial dysfunction play pivotal roles in promoting neurodegeneration, Cdk5 could be a viable therapeutic target for AD.

α-smooth muscle actin is not a marker of fibrogenic cell activity in skeletal muscle fibrosis.

α-Smooth muscle actin (α-SMA) is used as a marker for a subset of activated fibrogenic cells, myofibroblasts, which are regarded as important effector cells of tissue fibrogenesis. We address whether α-SMA-expressing myofibroblasts are detectable in fibrotic muscles of mdx5cv mice, a mouse model for Duchenne muscular dystrophy (DMD), and whether the α-SMA expression correlates with the fibrogenic function of intramuscular fibrogenic cells. α-SMA immunostaining signal was not detected in collagen I (GFP)-expressing cells in fibrotic muscles of ColI-GFP/mdx5cv mice, but it was readily detected in smooth muscle cells lining intramuscular blood vessel walls. α-SMA expression was detected by quantitative RT-PCR and Western blot in fibrogenic cells sorted from diaphragm and quadriceps muscles of the ColI-GFP/mdx5cv mice. Consistent with the more severe fibrosis in the ColI-GFP/mdx5cv diaphragm, the fibrogenic cells in the diaphragm exerted a stronger fibrogenic function than the fibrogenic cells in the quadriceps as gauged by their extracellular matrix gene expression. However, both gene and protein expression of α-SMA was lower in the diaphragm fibrogenic cells than in the quadriceps fibrogenic cells in the ColI-GFP/mdx5cv mice. We conclude that myofibroblasts are present in fibrotic skeletal muscles, but their expression of α-SMA is not detectable by immunostaining. The level of α-SMA expression by intramuscular fibrogenic cells does not correlate positively with the level of collagen gene expression or the severity of skeletal muscle fibrosis in the mdx5cv mice. α-SMA is not a functional marker of fibrogenic cells in skeletal muscle fibrosis associated with muscular dystrophy.

Nuclear envelope dispersion triggered by deregulated Cdk5 precedes neuronal death.

Nuclear fragmentation is a common feature in many neurodegenerative diseases, including Alzheimer's disease (AD). In this study, we show that nuclear lamina dispersion is an early and irreversible trigger for cell death initiated by deregulated Cdk5, rather than a consequence of apoptosis. Cyclin-dependent kinase 5 (Cdk5) activity is significantly increased in AD and contributes to all three hallmarks: neurotoxic amyloid-ß (Aß), neurofibrillary tangles (NFT), and extensive cell death. Using Aß and glutamate as the neurotoxic stimuli, we show that deregulated Cdk5 induces nuclear lamina dispersion by direct phosphorylation of lamin A and lamin B1 in neuronal cells and primary cortical neurons. Phosphorylation-resistant mutants of lamins confer resistance to nuclear dispersion and cell death on neurotoxic stimulation, highlighting this as a major mechanism for neuronal death. Rapid alteration of lamin localization pattern and nuclear membrane change are further supported by in vivo data using an AD mouse model. After p25 induction, the pattern of lamin localization was significantly altered, preceding neuronal death, suggesting that it is an early pathological event in p25-inducible transgenic mice. Importantly, lamin dispersion is coupled with Cdk5 nuclear localization, which is highly neurotoxic. Inhibition of nuclear dispersion rescues neuronal cells from cell death, underscoring the significance of this event to Cdk5-mediated neurotoxicity.

Direct and indirect roles of cyclin-dependent kinase 5 as an upstream regulator in the c-Jun NH2-terminal kinase cascade: relevance to neurotoxic insults in Alzheimer's disease.

Significant increase in JNK, c-Jun, and Cdk5 activities are reported in Alzheimer's disease (AD). Inhibition of c-Jun prevents neuronal cell death in in vivo AD models, highlighting it as a major JNK effector. Both JNK and Cdk5 promote neurodegeneration upon deregulation; however, Cdk5 has not been mechanistically linked to JNK or c-Jun. This study presents the first mechanism showing Cdk5 as a major regulator of the JNK cascade. Deregulated Cdk5 induces biphasic activation of JNK pathway. The first phase revealed c-Jun as a direct substrate of Cdk5, whose activation is independent of reactive oxygen species (ROS) and JNK. In the second phase, Cdk5 activates c-Jun via ROS-mediated activation of JNK. Rapid c-Jun activation is supported by in vivo data showing c-Jun phosphorylation in cerebral cortex upon p25 induction in transgenic mice. Cdk5-mediated biphasic activation of c-Jun highlights c-Jun, rather than JNK, as an important therapeutic target, which was confirmed in neuronal cells. Finally, Cdk5 inhibition endows superior protection against neurotoxicity, suggesting that Cdk5 is a preferable therapeutic target for AD relative to JNK and c-Jun.

Novel genetic tools reveal Cdk5's major role in Golgi fragmentation in Alzheimer's disease.

Golgi fragmentation is a common feature in multiple neurodegenerative diseases; however, the precise mechanism that causes fragmentation remains obscure. A potential link between Cdk5 and Golgi fragmentation in Alzheimer's disease (AD) was investigated in this study. Because Golgi is physiologically fragmented during mitosis by Cdc2 kinase and current Cdk5-specific chemical inhibitors target Cdc2 as well, development of novel tools to modulate Cdk5 activity was essential. These enzyme modulators, created by fusing TAT sequence to Cdk5 activators and an inhibitor peptide, enable specific activation and inhibition of Cdk5 activity with high temporal control. These genetic tools revealed a major role of Cdk5 in Golgi fragmentation upon beta-amyloid and glutamate stimulation in differentiated neuronal cells and primary neurons. A crucial role of Cdk5 was further confirmed when Cdk5 activation alone resulted in robust Golgi disassembly. The underlying mechanism was unraveled using a chemical genetic screen, which yielded cis-Golgi matrix protein GM130 as a novel substrate of Cdk5. Identification of the Cdk5 phosphorylation site on GM130 suggested a mechanism by which Cdk5 may cause Golgi fragmentation upon deregulation in AD. As Cdk5 is activated in several neurodegenerative diseases where Golgi disassembly also occurs, this may be a common mechanism among multiple disorders.

Blue light negatively regulates the sexual filamentation via the Cwc1 and Cwc2 proteins in Cryptococcus neoformans.

Cryptococcus neoformans is a heterothallic basidiomycetous yeast that primarily infects immunocompromised individuals. Dikaryotic hyphae resulting from the fusion of the MATa and MATalpha mating type strains represent the filamentous stage in the sexual life cycle of C. neoformans. In this study we demonstrate that the production of dikaryotic filaments is inhibited by blue light. To study blue light photoresponse in C. neoformans, we have identified and characterized two genes, CWC1 and CWC2, which are homologous to Neurospora crassa wc-1 and wc-2 genes. Conserved domain analyses indicate that the functions of Cwc1 and Cwc2 proteins may be evolutionally conserved. To dissect their roles in the light response, the CWC1 gene deletion mutants are created in both mating type strains. Mating filamentation in the bilateral cross of cwc1 MATa and MATalpha strains is not sensitive to light. The results indicate that Cwc1 may be an essential regulator of light responses in C. neoformans. Furthermore, overexpression of the CWC1 or CWC2 gene requires light activation to inhibit sexual filamentation, suggesting both genes may function together in the early step of blue light signalling. Taken together, our findings illustrate blue light negatively regulates the sexual filamentation via the Cwc1 and Cwc2 proteins in C. neoformans.