Scleraxis is a basic helix-loop-helix transcription factor that plays a central role in promoting tenocyte proliferation and matrix synthesis during embryonic tendon development. However, the role of scleraxis in the growth and adaptation of adult tendons is not known. We hypothesized that scleraxis is required for tendon growth in response to mechanical loading, and that scleraxis promotes the specification of progenitor cells into tenocytes. We conditionally deleted scleraxis in adult mice using a tamoxifen-inducible Cre-recombinase expressed from the Rosa26 locus (ScxΔ), and then induced tendon growth in Scx+ and ScxΔ adult mice via plantaris tendon mechanical overload. Compared to the wild type Scx+ group, ScxΔ mice demonstrated blunted tendon growth. Transcriptional and proteomic analyses revealed significant reductions in cell proliferation, protein synthesis, and extracellular matrix genes and proteins. Our results indicate that scleraxis is required for mechanically-stimulated adult tendon growth by causing the commitment of CD146+ pericytes into the tenogenic lineage, and by promoting the initial expansion of newly committed tenocytes and the production of extracellular matrix proteins.
Jonathan P. Gumucio, Martin M. Schonk, Yalda A. Kharaz, Eithne Comerford, Christopher L. Mendias
In pulmonary hypertension and certain forms of congenital heart disease, ventricular pressure overload manifests at birth and is an obligate hemodynamic abnormality that stimulates myocardial fibrosis which leads to ventricular dysfunction and poor clinical outcomes. Thus, an attractive strategy is to attenuate the myocardial fibrosis to help preserve ventricular function. Here, by analyzing RNA-sequencing databases and comparing the transcript and protein levels of fibrillar collagen in wild-type and global knockout mice, we found that SLIT3 was predominantly present in fibrillar collagen-producing cells and that SLIT3 deficiency attenuated collagen production in the heart and other non-neuronal tissues. We then performed transverse aortic constriction or pulmonary artery banding in wild-type and knockout mice to induce left and right ventricular pressure overload, respectively. We discovered that SLIT3 deficiency abrogates fibrotic and hypertrophic changes and promotes long-term ventricular function and overall survival in both left and right ventricular pressure overload. Furthermore, we found that SLIT3 stimulated fibroblast activity and fibrillar collagen production, which coincided with the transcription and nuclear localization of the mechanotransducer YAP1. These results indicate that SLIT3 is important for regulating fibroblast activity and fibrillar collagen synthesis in an autocrine manner, making it a potential therapeutic target for fibrotic diseases, especially myocardial fibrosis and adverse remodeling induced by persistent afterload elevation.
Lianghui Gong, Shuyun Wang, Li Shen, Catherine Liu, Mena Shenouda, Baolei Li, Xiaoxiao Liu, John Shaw, Alan Wineman, Yifeng Yang, Dingding Xiong, Anne Eichmann, Sylvia M. Evans, Stephen J. Weiss, Ming-Sing Si
The Wnt/beta-catenin signaling pathway plays an important role in renal development and is re-expressed in the injured kidney and other organs. Beta-catenin signaling is protective in acute kidney injury (AKI) through actions on the proximal tubule, but the current dogma is that Wnt/beta-catenin signaling promotes fibrosis and development of chronic kidney disease (CKD). As the role of proximal tubular beta-catenin signaling in CKD remains unclear, we genetically stabilized (i.e. activated) beta-catenin specifically in murine proximal tubules. Mice with increased tubular beta-catenin signaling were protected in two different murine models of AKI to CKD progression. Oxidative stress, a common feature of CKD, reduced the conventional TCF/LEF-dependent beta-catenin signaling and augmented FoxO3-dependent activity in proximal tubule cells in vitro and in vivo. The protective effect of proximal tubular beta-catenin in renal injury required the presence of FoxO3 in vivo. Furthermore, we identified cystathionine gamma-lyase (CSE) as a novel transcriptional target of beta-catenin/FoxO3 interactions in the proximal tubule. Thus, our studies overturn the conventional dogma about beta-catenin signaling and CKD by showing a protective effect of proximal tubule beta-catenin in CKD and identified a new transcriptional target of beta-catenin/FoxO3 signaling that has therapeutic potential for CKD.
Stellor Nlandu-Khodo, Yosuke Osaki, Lauren Scarfe, Hai-chun Yang, Melanie Phillips-Mignemi, Jane Tonello, Kenyi Saito-Diaz, Surekha Neelisetty, Alla V. Ivanova, Tessa Huffstater, Robert S. McMahon, Makoto M. Taketo, Mark deCaestecker, Balakuntalam S. Kasinath, Raymond C. Harris, Ethan Lee, Leslie Gewin
The IL1RL1 (ST2) gene locus is robustly associated with asthma; however, the contribution of single nucleotide polymorphisms (SNPs) in this locus to specific asthma subtypes and the functional mechanisms underlying these associations remain to be defined. We tested for association between IL1RL1 region SNPs and characteristics of asthma as defined by clinical and immunological measures and addressed functional effects of these genetic variants in lung tissue and airway epithelium. Utilizing 4 independent cohorts (Lifelines, Dutch Asthma GWAS [DAG], Genetics of Asthma Severity and Phenotypes [GASP], and Manchester Asthma and Allergy Study [MAAS]) and resequencing data, we identified 3 key signals associated with asthma features. Investigations in lung tissue and primary bronchial epithelial cells identified context-dependent relationships between the signals and IL1RL1 mRNA and soluble protein expression. This was also observed for asthma-associated IL1RL1 nonsynonymous coding TIR domain SNPs. Bronchial epithelial cell cultures from asthma patients, exposed to exacerbation-relevant stimulations, revealed modulatory effects for all 4 signals on IL1RL1 mRNA and/or protein expression, suggesting SNP-environment interactions. The IL1RL1 TIR signaling domain haplotype affected IL-33–driven NF-κB signaling, while not interfering with TLR signaling. In summary, we identify that IL1RL1 genetic signals potentially contribute to severe and eosinophilic phenotypes in asthma, as well as provide initial mechanistic insight, including genetic regulation of IL1RL1 isoform expression and receptor signaling.
Michael A. Portelli, F. Nicole Dijk, Maria E. Ketelaar, Nick Shrine, Jenny Hankinson, Sangita Bhaker, Néomi S. Grotenboer, Ma’en Obeidat, Amanda P. Henry, Charlotte K. Billington, Dominick Shaw, Simon R. Johnson, Zara E.K. Pogson, Andrew Fogarty, Tricia M. McKeever, David C. Nickle, Yohan Bossé, Maarten van den Berge, Alen Faiz, Sharon Brouwer, Judith M. Vonk, Paul de Vos, Corry-Anke Brandsma, Cornelis J. Vermeulen, Amisha Singapuri, Liam G. Heaney, Adel H. Mansur, Rekha Chaudhuri, Neil C. Thomson, John W. Holloway, Gabrielle A. Lockett, Peter H. Howarth, Robert Niven, Angela Simpson, John D. Blakey, Martin D. Tobin, Dirkje S. Postma, Ian P. Hall, Louise V. Wain, Martijn C. Nawijn, Christopher E. Brightling, Gerard H. Koppelman, Ian Sayers
Advanced colorectal cancer (CRC) is often accompanied by development of liver metastases (LMs) and skeletal muscle (SkM) wasting, i.e. cachexia. Despite plaguing the majority of CRC patients, cachexia remains unresolved. By using mice subcutaneously (C26) or intrasplenically injected with C26 tumor cells to mimic hepatic dissemination of cancer cells (mC26), here we aimed to further characterize functional, molecular and metabolic effects on SkM and examine whether LMs exacerbate CRC-induced cachexia. C26-derived LMs were associated with progressive loss of body weight, as well as with significant reductions in SkM size and strength, in line with reduced phosphorylation of markers of protein anabolism and enhanced protein catabolism. mC26 hosts showed prevalence of fibers with glycolytic metabolism and enhanced lipid accumulation, consistent with abnormalities of mitochondrial homeostasis and energy metabolism. In a comparison with mice bearing subcutaneous C26, cachexia appeared exacerbated in the mC26 hosts, as also supported by differentially expressed pathways within SkM. Overall, our model recapitulates the cachectic phenotype of metastatic CRC and reveals that formation of LMs resulting from CRC exacerbate cancer-induced SkM wasting by promoting differential gene expression signatures.
Joshua R. Huot, Leah J. Novinger, Fabrizio Pin, Ashok Narasimhan, Teresa A. Zimmers, Thomas M. O'Connell, Andrea Bonetto
BK channels are expressed in intercalated (ICs) and principal (PCs) cells in the cortical collecting duct (CCD) of the mammalian kidney and have been proposed to be responsible for flow-induced K+ secretion (FIKS) and K+ adaptation. To examine the IC-specific role of BK channels, we generated a mouse with targeted disruption of the pore-forming BK alpha subunit (BKα) in ICs (IC-BKα-KO). Whole cell charybdotoxin (ChTX)-sensitive K+ currents were readily detected in control ICs, but largely absent in ICs of IC-BKα-KO mice. When placed on a high K+ (HK) diet for 13 days, blood [K+] was significantly greater in IC-BKα-KO mice vs. controls in males only, although urinary K+ excretion rates following isotonic volume expansion were similar in males and females. FIKS was present in microperfused CCDs isolated from controls, but was absent in IC-BKα-KO CCDs of both sexes. Also, flow-stimulated ENaC-mediated Na+ absorption was greater in CCDs from female IC-BKα-KO mice than in CCDs from males. Our results confirm a critical role of IC BK channels in FIKS. Sex contributes to the capacity for adaptation to a HK diet in IC-BKα-KO mice.
Rolando Carrisoza-Gaytan, Evan C. Ray, Daniel Flores, Allison L. Marciszyn, Peng Wu, Leah Liu, Arohan R. Subramanya, WenHui Wang, Shaohu Sheng, Lubika J. Nkashama, Jingxin Chen, Edwin K. Jackson, Stephanie M. Mutchler, Szilvia Heja, Donald E. Kohan, Lisa M. Satlin, Thomas R. Kleyman
Hydrocephalus is characterized by abnormal accumulation of cerebrospinal fluid (CSF) in the ventricular cavity. The circulation of CSF in brain ventricles is controlled by the coordinated beating of motile cilia at the surface of ependymal cells (ECs). Here we show that MT1-MMP is highly expressed in olfactory bulb, rostral migratory stream, and ventricular system. Mice deficient for Membrane type-1-MMP (MT1-MMP) develop typical phenotypes observed in hydrocephalus such as dome-shaped skull, dilated ventricles, corpus callosum agenesis and astrocyte hypertrophy during the first two weeks of postnatal development. MT1-MMP deficient mice exhibits reduced and disorganized motile cilia with the impaired maturation of ECs, leading to abnormal CSF flow. Consistent with the defects in motile cilia morphogenesis, the expressions of pro-multiciliogenic genes are significantly decreased with a concomitant hyper-activation of Notch signaling in the wall of lateral ventricles in Mmp14-/- brains. Inhibition of Notch signaling by γ-secretase inhibitor restores ciliogenesis in Mmp14-/- ECs. Taken together, these data suggest that MT1-MMP is required for ciliogenesis and ependymal cell maturation by suppressing Notch signaling during early brain development. Our findings implicate that MT1-MMP is critical for early brain development and loss of MT1-MMP activity gives rise to hydrocephalus.
Zhixin Jiang, Jin Zhou, Xin Qin, Huiling Zheng, Bo Gao, Xin-guang Liu, Guoxiang Jin, Zhongjun Zhou
Familial Hypocalciuric Hypercalcemia (FHH) is a genetic condition associated with hypocalciuria, hypercalcemia and in some cases inappropriately high levels of circulating parathyroid hormone (PTH). FHH is associated with inactivating mutations in CaSR encoding the Ca2+ sensing receptor (CaSR), a G protein coupled receptor (GPCR) and GNA11 encoding G protein subunit alpha 11 (Gα11), implicating defective GPCR signaling as the root pathophysiology for FHH. However, the downstream mechanism by which CaSR activation inhibits PTH production/secretion is incompletely understood. Here, we show that mice lacking the transient receptor potential canonical channel 1 (TRPC1) develop chronic hypercalcemia, hypocalciuria, and elevated PTH levels mimicking human FHH. Ex vivo and in vitro studies reveal that TRPC1 serves a necessary and sufficient mediator to suppress PTH secretion from parathyroid glands (PTG) downstream of CaSR in response to high extracellular Ca2+ concentration. Gα11 physically interacts with both the N- and C-termini of TRPC1 and enhances CaSR-induced TRPC1 activity in transfected cells. These data identify TRPC1-mediated Ca2+ signaling as an essential component of the cellular apparatus controlling PTH secretion in the PTG downstream of CaSR.
Marta Onopiuk, Bonnie Eby, Vasyl Nesin, Peter Ngo, Megan Lerner, Caroline M. Gorvin, Victoria J. Stokes, Rajesh V. Thakker, Maria Luisa Brandi, Wenhan Chang, Mary Beth Humphrey, Leonidas Tsiokas, Kai Lau
Duchenne muscular dystrophy (DMD) is a chronic muscle disease characterized by poor myogenesis and replacement of muscle by extracellular matrix. Despite the shared genetic basis, severity of these deficits varies among patients. One source of these variations is the genetic modifier that leads to increased TGF-β activity. While anti–TGF-β therapies are being developed to target muscle fibrosis, their effect on the myogenic deficit is underexplored. Our analysis of in vivo myogenesis in mild (C57BL/10ScSn-mdx/J and C57BL/6J-mdxΔ52) and severe DBA/2J-mdx (D2-mdx) dystrophic models reveals no defects in developmental myogenesis in these mice. However, muscle damage at the onset of disease pathology, or by experimental injury, drives up TGF-β activity in the severe, but not in the mild, dystrophic models. Increased TGF-β activity is accompanied by increased accumulation of fibroadipogenic progenitors (FAPs) leading to fibro-calcification of muscle, together with failure of regenerative myogenesis. Inhibition of TGF-β signaling reduces muscle degeneration by blocking FAP accumulation without rescuing regenerative myogenesis. These findings provide in vivo evidence of early-stage deficit in regenerative myogenesis in D2-mdx mice and implicates TGF-β as a major component of a pathogenic positive feedback loop in this model, identifying this feedback loop as a therapeutic target.
Davi A.G. Mázala, James S. Novak, Marshall W. Hogarth, Marie Nearing, Prabhat Adusumalli, Christopher B. Tully, Nayab F. Habib, Heather Gordish-Dressman, Yi-Wen Chen, Jyoti K. Jaiswal, Terence A. Partridge
Abnormal wound repair has been observed in the airway epithelium of patients with chronic respiratory diseases including asthma. Therapies focusing on repairing vulnerable airways, particularly in early life, present an extremely novel treatment strategy. We report defective lower airway epithelial cell repair to strongly associate with common pre-school and school-aged wheezing phenotypes, characterised by aberrant migration patterns and reduced α5β1 integrin expression. Next generation sequencing identified the PI3K/Akt pathway as the top upstream transcriptional regulator of α5β1 integrin, where Akt activation enhanced repair and α5β1 integrin expression in primary cultures from children with wheeze. Conversely, inhibition of PI3K/Akt signaling in primary cultures from children without wheeze reduced α5β1 expression and attenuated repair. Importantly, the FDA-approved drug celecoxib, and its non-COX2-inhibiting analogue dimethyl-celecoxib, stimulated the PI3K/Akt-integrin α5β1 axis and restored airway epithelial repair in cells from children with wheeze. When compared with published clinical datasets the identified transcriptomic signature was also associated with viral-induced wheeze exacerbations highlighting the clinical potential of such therapy. Collectively, these results identify airway epithelial restitution via targeting the PI3K/Akt-integrin axis as a novel therapeutic avenue for childhood wheeze and asthma. We propose that the next step in the therapeutic development process should be a proof-of-concept clinical trial since relevant animal models to test the crucial underlying premise are unavailable.
Thomas Iosifidis, Erika N. Sutanto, Alysia Buckley, Laura A. Coleman, Erin E. Gill, Amy H. Lee, Kak-Ming Ling, Jessica Hillas, Kevin Looi, Luke W. Garratt, Kelly M. Martinovich, Nicole C. Shaw, Samuel T. Montgomery, Elizabeth Kicic-Starcevich, Yuliya V. Karpievitch, Peter Le Souef, Ingrid A. Laing, Shyan Vijayasekaran, Francis J. Lannigan, Paul J. Rigby, Robert E.W. Hancock, Darryl Knight, Stephen M. Stick, Anthony Kicic, on behalf of WAERP, on behalf of AusREC
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