Huntingtin interacting protein 1 related (Hip1r) is an F-actin– and clathrin-binding protein involved in vesicular trafficking. In this study, we demonstrate that Hip1r is abundantly expressed in the gastric parietal cell, predominantly localizing with F-actin to canalicular membranes. Hip1r may provide a critical function in vivo, as demonstrated by extensive changes to parietal cells and the gastric epithelium in Hip1r-deficient mice. Electron microscopy revealed abnormal apical canalicular membranes and loss of tubulovesicles in mutant parietal cells, suggesting that Hip1r is necessary for the normal trafficking of these secretory membranes. Accordingly, acid secretory dynamics were altered in mutant parietal cells, with enhanced activation and acid trapping, as measured in isolated gastric glands. At the whole-organ level, gastric acidity was reduced in Hip1r-deficient mice, and the gastric mucosa was grossly transformed, with fewer parietal cells due to enhanced apoptotic cell death and glandular hypertrophy associated with cellular transformation. Hip1r-deficient mice had increased expression of the gastric growth factor gastrin, and mice mutant for both gastrin and Hip1r exhibited normalization of both proliferation and gland height. Taken together, these studies demonstrate that Hip1r plays a significant role in gastric physiology, mucosal architecture, and secretory membrane dynamics in parietal cells.
Renu N. Jain, Asma A. Al-Menhali, Theresa M. Keeley, Jianhua Ren, Mohammed El-Zaatari, Xunsheng Chen, Juanita L. Merchant, Theodora S. Ross, Catherine S. Chew, Linda C. Samuelson
Elevated intraocular pressure (IOP) is the principal risk factor for glaucoma and results from excessive impedance of the fluid outflow from the eye. This abnormality likely originates from outflow pathway tissues such as the trabecular meshwork (TM), but the associated molecular etiology is poorly understood. We discovered what we believe to be a novel role for secreted frizzled-related protein-1 (sFRP-1), an antagonist of Wnt signaling, in regulating IOP. sFRP1 was overexpressed in human glaucomatous TM cells. Genes involved in the Wnt signaling pathway were expressed in cultured TM cells and human TM tissues. Addition of recombinant sFRP-1 to ex vivo perfusion-cultured human eyes decreased outflow facility, concomitant with reduced levels of β-catenin, the Wnt signaling mediator, in the TM. Intravitreal injection of an adenoviral vector encoding sFRP1 in mice produced a titer-dependent increase in IOP. Five days after vector injection, IOP increased 2 fold, which was significantly reduced by topical ocular administration of an inhibitor of a downstream suppressor of Wnt signaling. Thus, these data indicate that increased expression of sFRP1 in the TM appears to be responsible for elevated IOP in glaucoma and restoring Wnt signaling in the TM may be a novel disease intervention strategy for treating glaucoma.
Wan-Heng Wang, Loretta G. McNatt, Iok-Hou Pang, J. Cameron Millar, Peggy E. Hellberg, Mark H. Hellberg, H. Thomas Steely, Jeffrey S. Rubin, John H. Fingert, Val C. Sheffield, Edwin M. Stone, Abbot F. Clark
ER stress can cause hepatic insulin resistance and steatosis. Increased VLDL secretion could protect the liver from ER stress–induced steatosis, but the effect of lipid-induced ER stress on the secretion of VLDL is unknown. To determine the effect of lipids on hepatic ER stress and VLDL secretion, we treated McA-RH7777 liver cells with free fatty acids. Prolonged exposure increased cell triglycerides, induced steatosis, and increased ER stress. Effects on apoB100 secretion, which is required for VLDL assembly, were parabolic, with moderate free fatty acid exposure increasing apoB100 secretion, while greater lipid loading inhibited apoB100 secretion. This decreased secretion at higher lipid levels was due to increased protein degradation through both proteasomal and nonproteasomal pathways and was dependent on the induction of ER stress. These findings were supported in vivo, where intravenous infusion of oleic acid (OA) in mice increased ER stress in a duration-dependent manner. apoB secretion was again parabolic, stimulated by moderate, but not prolonged, OA infusion. Inhibition of ER stress was able to restore OA-stimulated apoB secretion after prolonged OA infusion. These results suggest that excessive ER stress in response to increased hepatic lipids may decrease the ability of the liver to secrete triglycerides by limiting apoB secretion, potentially worsening steatosis.
Tsuguhito Ota, Constance Gayet, Henry N. Ginsberg
Progressive pulmonary disease and infections with Pseudomonas aeruginosa remain an intractable problem in cystic fibrosis (CF). At the cellular level, CF is characterized by organellar hyperacidification, which results in altered protein and lipid glycosylation. Altered pH of the trans-Golgi network (TGN) may further disrupt the protein processing and packaging that occurs in this organelle. Here we measured activity of the major TGN endoprotease furin and demonstrated a marked upregulation in human CF cells. Increased furin activity was linked to elevated production in CF of the immunosuppressive and tissue remodeling cytokine TGF-β and its downstream effects, including macrophage deactivation and augmented collagen secretion by epithelial cells. As furin is responsible for the proteolytic processing of a range of endogenous and exogenous substrates including growth factors and bacterial toxins, we determined that elevated furin-dependent activation of exotoxin A caused increased cell death in CF respiratory epithelial cells compared with genetically matched CF transmembrane conductance regulator–corrected cells. Thus elevated furin levels in CF respiratory epithelial cells contributes to bacterial toxin–induced cell death, fibrosis, and local immunosuppression. These data suggest that the use of furin inhibitors may represent a strategy for pharmacotherapy in CF.
Wojciech Ornatowski, Jens F. Poschet, Elizabeth Perkett, Jennifer L. Taylor-Cousar, Vojo Deretic
Charuhas V. Thakar, Kamyar Zahedi, Monica P. Revelo, Zhaohui Wang, Charles E. Burnham, Sharon Barone, Shannon Bevans, Alex B. Lentsch, Hamid Rabb, Manoocher Soleimani
IL-13 dysregulation plays a critical role in the pathogenesis of a variety of inflammatory and remodeling diseases. In these settings, STAT6 is believed to be the canonical signaling molecule mediating the tissue effects of IL-13. Signaling cascades involving MAPKs have been linked to inflammation and remodeling. We hypothesized that MAPKs play critical roles in effector responses induced by IL-13 in the lung. We found that Tg IL-13 expression in the lung led to potent activation of ERK1/2 but not JNK1/2 or p38. ERK1/2 activation also occurred in mice with null mutations of STAT6. Systemic administration of the MAPK/ERK kinase 1 (MEK1) inhibitor PD98059 or use of Tg mice in which a dominant-negative MEK1 construct was expressed inhibited IL-13–induced inflammation and alveolar remodeling. There were associated decreases in IL-13–induced chemokines (MIP-1α/CCL-3, MIP-1β/CCL-4, MIP-2/CXCL-1, RANTES/CCL-5), MMP-2, -9, -12, and -14, and cathepsin B and increased levels of α1-antitrypsin. IL-13–induced tissue and molecular responses were noted that were equally and differentially dependent on ERK1/2 and STAT6 signaling. Thus, ERK1/2 is activated by IL-13 in the lung in a STAT6-independent manner where it contributes to IL-13–induced inflammation and remodeling and is required for optimal IL-13 stimulation of specific chemokines and proteases as well as the inhibition of specific antiproteases. ERK1/2 regulators may be useful in the treatment of IL-13–induced diseases and disorders.
Patty J. Lee, Xuchen Zhang, Peiying Shan, Bing Ma, Chun Geun Lee, Robert J. Homer, Zhou Zhu, Mercedes Rincon, Brooke T. Mossman, Jack A. Elias
Thrombospondin 1 (TSP-1) is a matricellular protein that inhibits angiogenesis and causes apoptosis in vivo and in vitro in several cancerous cells and tissues. Here we identify TSP-1 as the molecule with the highest induction level at 3 hours of IR injury in rat and mouse kidneys subjected to ischemia/reperfusion (IR) injury using the DNA microarray approach. Northern hybridizations demonstrated that TSP-1 expression was undetectable at baseline, induced at 3 and 12 hours, and returned to baseline levels at 48 hours of reperfusion. Immunocytochemical staining identified the injured proximal tubules as the predominant sites of expression of TSP-1 in IR injury and showed colocalization of TSP-1 with activated caspase-3. Addition of purified TSP-1 to normal kidney proximal tubule cells or cells subjected to ATP depletion in vitro induced injury as demonstrated by cytochrome c immunocytochemical staining and caspase-3 activity. The deleterious role of TSP-1 in ischemic kidney injury was demonstrated directly in TSP-1 null mice, which showed significant protection against IR injury–induced renal failure and tubular damage. We propose that TSP-1 is a novel regulator of ischemic damage in the kidney and may play an important role in the pathophysiology of ischemic kidney failure.
Charuhas V. Thakar, Kamyar Zahedi, Monica P. Revelo, Zhaohui Wang, Charles E. Burnham, Sharon Barone, Shannon Bevans, Alex B. Lentsch, Hamid Rabb, Manoocher Soleimani
Hemin upregulates heme oxygenase-1 (HO-1), a stress-induced enzyme implicated in protection from a variety of injuries while its related isoform HO-2 is constitutively expressed. The role of hemin or HO-1 in the pancreas and their potential modulation of pancreatic injury are unknown. We show that HO-1 is induced in pancreatitis caused by caerulein and more prominently in severe pancreatitis caused by feeding a choline-deficient diet (CDD). Intraperitoneal hemin administration dramatically increases peritoneal and pancreas macrophages that overexpress HO-1 in association with pancreatic induction of the chemoattractants monocyte chemotactic protein-1 and macrophage inflammatory protein-1α but not RANTES or macrophage inflammatory protein-2. Hemin administration before CDD feeding protected 8 of 8 mice from lethality while 7 of 16 controls died. Protection is mediated by HO-1–overexpressing macrophages since hemin-primed macrophages home to the pancreas after transfer to naive mice and protect from CDD-induced pancreatitis. Suppression of hemin-primed peritoneal cell HO-1 using HO-1–specific small interfering RNA prior to cell transfer abolishes protection from CDD-induced pancreatitis. Similarly, hemin pretreatment in caerulein-induced pancreatitis reduces serum amylase and lipase, decreases pancreatic trypsin generation, and protects from lung injury. Therefore, hemin-like compounds or hemin-activated macrophages may offer novel therapeutic approaches for preventing acute pancreatitis and its pulmonary complication via upregulation of HO-1.
Ikuo Nakamichi, Aida Habtezion, Bihui Zhong, Christopher H. Contag, Eugene C. Butcher, M. Bishr Omary
The umbrella cells that line the bladder are mechanosensitive, and bladder filling increases the apical surface area of these cells; however, the upstream signals that regulate this process are unknown. Increased pressure stimulated ATP release from the isolated uroepithelium of rabbit bladders, which was blocked by inhibitors of vesicular transport, connexin hemichannels, ABC protein family members, and nucleoside transporters. Pressure-induced increases in membrane capacitance (a measure of apical plasma membrane surface area where 1 μF ≈ 1 cm2) were inhibited by the serosal, but not mucosal, addition of apyrase or the purinergic receptor antagonist PPADS. Upon addition of purinergic receptor agonists, increased capacitance was observed even in the absence of pressure. Moreover, knockout mice lacking expression of P2X2 and/or P2X3 receptors failed to show increases in apical surface area when exposed to hydrostatic pressure. Treatments that prevented release of Ca2+ from intracellular stores or activation of PKA blocked ATPγS-stimulated changes in capacitance. These results indicate that increased hydrostatic pressure stimulates release of ATP from the uroepithelium and that upon binding to P2X and possibly P2Y receptors on the umbrella cell, downstream Ca2+ and PKA second messenger cascades may act to stimulate membrane insertion at the apical pole of these cells.
Edward C.Y. Wang, Jey-Myung Lee, Wily G. Ruiz, Elena M. Balestreire, Maximilian von Bodungen, Stacey Barrick, Debra A. Cockayne, Lori A. Birder, Gerard Apodaca
Synaptopodin is the founding member of a novel class of proline-rich actin-associated proteins highly expressed in telencephalic dendrites and renal podocytes. Synaptopodin-deficient (synpo–/–) mice lack the dendritic spine apparatus and display impaired activity-dependent long-term synaptic plasticity. In contrast, the ultrastructure of podocytes in synpo–/– mice is normal. Here we show that synpo–/– mice display impaired recovery from protamine sulfate–induced podocyte foot process (FP) effacement and LPS-induced nephrotic syndrome. Similarly, synpo–/– podocytes show impaired actin filament reformation in vitro. We further demonstrate that synaptopodin exists in 3 isoforms, neuronal Synpo-short (685 AA), renal Synpo-long (903 AA), and Synpo-T (181 AA). The C terminus of Synpo-long is identical to that of Synpo-T. All 3 isoforms specifically interact with α-actinin and elongate α-actinin–induced actin filaments. synpo–/– mice lack Synpo-short and Synpo-long expression but show an upregulation of Synpo-T protein expression in podocytes, though not in the brain. Gene silencing of Synpo-T abrogates stress-fiber formation in synpo–/– podocytes, demonstrating that Synpo-T serves as a backup for Synpo-long in synpo–/– podocytes. In concert, synaptopodin regulates the actin-bundling activity of α-actinin in highly dynamic cell compartments, such as podocyte FPs and the dendritic spine apparatus.
Katsuhiko Asanuma, Kwanghee Kim, Jun Oh, Laura Giardino, Sophie Chabanis, Christian Faul, Jochen Reiser, Peter Mundel
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