Novel Treatment of Chronic Graft-Versus-Host Disease in Mice Using the ER Stress Reducer 4-Phenylbutyric Acid.
ABSTRACT: Chronic graft-versus-host disease (cGVHD) is a notorious complication of allogeneic hematopoietic stem cell transplantation and causes disabling systemic inflammation and fibrosis. In this novel study, we focused on a relationship between endoplasmic reticulum (ER) stress and cGVHD, and aimed to create effective treatment of cGVHD. A series of experiments were conducted using a mouse model of cGVHD. Our data suggested (1) that ER stress was elevated in organs affected by cGVHD and (2) that 4-phenylbutyric acid (PBA) could reduce cGVHD-induced ER stress and thereby alleviate systemic inflammation and fibrosis. Because fibroblasts are thought to be implicated in cGVHD-elicited fibrosis and because macrophages are reported to play a role in the development of cGVHD, we investigated cGVHD-triggered ER stress in fibroblasts and macrophages. Our investigation demonstrated (1) that indicators for ER stress and activation markers for fibroblasts were elevated in cGVHD-affected lacrimal gland fibroblasts and (2) that they could be reduced by PBA. Our work also indicated that splenic macrophages from PBA-dosed mice exhibited the lower levels of ER stress and M2 macrophage markers than those from cGVHD-affected mice. Collectively, this study suggests that the reduction of ER stress utilizing PBA can be a clinically translatable method to treat systemic cGVHD.
Project description:Chronic graft-versus-host disease (cGVHD) is a severe complication of allogeneic haematopoietic stem cell transplantation. There is a growing understanding of cGVHD, and several effective therapies for cGVHD have been reported. However, pancreatic cGVHD is a potentially untapped study field. Our thought-provoking study using a mouse model of cGVHD suggested that the pancreas could be impaired by cGVHD-induced inflammation and fibrosis and that endoplasmic reticulum (ER) stress was augmented in the pancreas affected by cGVHD. These findings urged us to treat pancreatic cGVHD through reduction of ER stress, and we used 4-phenylbutyric acid (PBA) as an ER stress reducer. A series of experiments has indicated that PBA can suppress cGVHD-elicited ER stress in the pancreas and accordingly alleviate pancreatic cGVHD. Furthermore, we focused on a correlation between epithelial to mesenchymal transition (EMT) and fibrosis in the cGVHD-affected pancreas, because EMT was conceivably implicated in various fibrosis-associated diseases. Our investigation has suggested that the expression of EMT markers was increased in the cGVHD-disordered pancreas and that it could be reduced by PBA. Taken together, we have provided a clue to elucidate the pathogenic process of pancreatic cGVHD and created a potentially effective treatment of this disease using the ER stress alleviator PBA.
Project description:An elevated level of endoplasmic reticulum (ER) stress is considered an aggravating factor for inflammatory bowel disease (IBD). To develop an ER-stress attenuator that is effective against colitis, 4-phenylbutyric acid (4-PBA), a chemical chaperone that alleviates ER stress, was conjugated with acidic amino acids to yield 4-PBA-glutamic acid (PBA-GA) and 4-PBA-aspartic acid (PBA-AA) conjugates. The PBA derivatives were converted to 4-PBA in the cecal contents, and the conversion was greater with PBA-GA than that with PBA-AA. After oral administration of PBA-GA (oral PBA-GA), up to 2.7 mM PBA was detected in the cecum, whereas 4-PBA was not detected in the blood, indicating that PBA-GA predominantly targeted the large intestine. In 2,4-dinitrobenzenesulfonic acid-induced colitis in rats, oral PBA-GA alleviated the damage and inflammation in the colon and substantially reduced the elevated levels of ER stress marker proteins in the inflamed colon. Moreover, PBA-GA was found to be as effective as the currently used anti-IBD drug, sulfasalazine. In conclusion, PBA-GA is a colon-targeted prodrug of 4-PBA and is effective against rat colitis probably via the attenuation of ER stress in the inflamed colon.
Project description:4-Phenylbutyric acid (4-PBA) is a chemical chaperone that eliminates the accumulation of unfolded proteins in the endoplasmic reticulum (ER). However, its chaperoning ability is often weak and unable to attenuate the unfolded protein response (UPR) in vitro or in vivo. To develop more potent chemical chaperones, we synthesized six analogues of 4-PBA and evaluated their pharmacological actions on the UPR.NRK-52E cells were treated with ER stress inducers (tunicamycin or thapsigargin) in the presence of each of the 4-PBA analogues; the suppressive effects of these analogues on the UPR were assessed using selective indicators for individual UPR pathways.2-POAA-OMe, 2-POAA-NO2 and 2-NOAA, but not others, suppressed the induction of ER stress markers GRP78 and CHOP. This suppressive effect was more potent than that of 4-PBA. Of the three major UPR branches, the IRE1 and ATF6 pathways were markedly blocked by these compounds, as indicated by suppression of XBP1 splicing, inhibition of UPRE and ERSE activation, and inhibition of JNK phosphorylation. Unexpectedly, however, these agents did not inhibit phosphorylation of PERK and eIF2? triggered by ER stress. These compounds dose-dependently inhibited the early activation of NF-?B in ER stress-exposed cells. 2-POAA-OMe and 2-POAA-NO2 also inhibited ER stress-induced phosphorylation of Akt.The 4-PBA analogues 2-POAA-OMe, 2-POAA-NO2 and 2-NOAA strongly inhibited activation of the IRE1 and ATF6 pathways and downstream pathogenic targets, including NF-?B and Akt, in ER stress-exposed cells. These compounds may be useful for therapeutic intervention in ER stress-related pathological conditions.
Project description:Endoplasmic reticulum (ER) stress is implicated in the pathophysiology of various cardiovascular diseases, but the role of ER stress in cardiac rupture and/or remodeling after myocardial infarction (MI) is still unclear. Here we investigated whether ER stress plays a major role for these processes in mice. We ligated the left coronary artery (LCA) without reperfusion in mice and administered either NaCl or 4-phenylbutyric acid (4-PBA, 20 mg/kg/d) intraperitoneally for 4 weeks. Cardiac rupture rates during the first week of MI were 37.5% and 18.2% in the control and 4-PBA groups, respectively. The extent of ventricular aneurysm and fibrosis was less, and the cardiac function better, in the 4-PBA group compared with the control group. The protein levels of ER stress markers in the heart tissues of the control group remained elevated during the entire 4-week period after MI, while pro-apoptotic proteins mainly increased in the early phase, and the pro-fibrotic proteins markedly increased in the late phase post MI; 4-PBA decreased all of these protein levels. In the primary cultured neonatal rat cardiomyocytes or fibroblasts, hypoxia (3% O2) increased the number of apoptotic cardiomyocytes and promoted the proliferation and migration of fibroblasts, all of which were attenuated by 4-PBA (0.5 mM). These findings indicate that MI induces ER stress and provokes cardiac apoptosis and fibrosis, culminating in cardiac rupture and remodeling, and that the attenuation of ER stress could be an effective therapeutic target to prevent post-MI complications.
Project description:BACKGROUND: The familial and sporadic forms of Alzheimer's disease (AD) have an identical pathology with a severe disparity in the time of onset . The pathological similarity suggests that epigenetic processes may phenocopy the Familial Alzheimer's disease (FAD) mutations within sporadic AD. Numerous groups have demonstrated that FAD mutations in presenilin result in 'loss of function' of gamma-secretase mediated APP cleavage , , , . Accordingly, ER stress is prominent within the pathologically impacted brain regions in AD patients  and is reported to inhibit APP trafficking through the secretory pathway , . As the maturation of APP and the cleaving secretases requires trafficking through the secretory pathway , , , we hypothesized that ER stress may block trafficking requisite for normal levels of APP cleavage and that the small molecular chaperone 4-phenylbutyrate (PBA) may rescue the proteolytic deficit. METHODOLOGY/PRINCIPAL FINDINGS: The APP-Gal4VP16/Gal4-reporter screen was stably incorporated into neuroblastoma cells in order to assay gamma-secretase mediated APP proteolysis under normal and pharmacologically induced ER stress conditions. Three unrelated pharmacological agents (tunicamycin, thapsigargin and brefeldin A) all repressed APP proteolysis in parallel with activation of unfolded protein response (UPR) signaling-a biochemical marker of ER stress. Co-treatment of the gamma-secretase reporter cells with PBA blocked the repressive effects of tunicamycin and thapsigargin upon APP proteolysis, UPR activation, and apoptosis. In unstressed cells, PBA stimulated gamma-secretase mediated cleavage of APP by 8-10 fold, in the absence of any significant effects upon amyloid production, by promoting APP trafficking through the secretory pathway and the stimulation of the non-pathogenic alpha/gamma-cleavage. CONCLUSIONS/SIGNIFICANCE: ER stress represses gamma-secretase mediated APP proteolysis, which replicates some of the proteolytic deficits associated with the FAD mutations. The small molecular chaperone PBA can reverse ER stress induced effects upon APP proteolysis, trafficking and cellular viability. Pharmaceutical agents, such as PBA, that stimulate alpha/gamma-cleavage of APP by modifying intracellular trafficking should be explored as AD therapeutics.
Project description:Objective:The integrity of cartilage depends on the correct synthesis of extracellular matrix (ECM) components. In case of insufficient folding of proteins in the endoplasmic reticulum (ER) of chondrocytes, ECM proteins aggregate, ER stress evolves, and the unfolded protein response (UPR) is initiated. By this mechanism, chondrocytes relieve the stress condition or initiate cell death by apoptosis. Especially persistent ER stress has emerged as a pathogenic mechanism in cartilage diseases, such as chondrodysplasias and osteoarthritis. As pharmacological intervention is not available yet, it is of great interest to understand cartilage ER stress in detail and to develop therapeutics to intervene. Methods:ERp57-deficient chondrocytes were generated by CRISPR/Cas9-induced KO. ER stress and autophagy were studied on mRNA and protein level as well as by transmission electron microscopy (TEM) in chondrocyte micromass or cartilage explant cultures of ERp57 KO mice. Thapsigargin (Tg), an inhibitor of the ER-residing Ca2+-ATPase, and 4-Phenylbutyric acid (4-PBA), a small molecular chemical chaperone, were applied to induce or inhibit ER stress. Results:Our data reveal that the loss of the protein disulfide isomerase ERp57 is sufficient to induce ER stress in chondrocytes. 4-PBA efficiently diffuses into cartilage explant cultures and diminishes excessive ER stress in chondrocytes dose dependently, no matter if it is induced by ERp57 KO or stimulation with Tg. Conclusion:ER-stress-related diseases have different sources; therefore, various targets for therapeutic treatment exist. In the future, 4-PBA may be used alone or in combination with other drugs for the treatment of ER-stress-related skeletal disorders in patients.
Project description:Renal tubulointerstitial fibrosis is the common and final pathologic change of kidney in end-stage renal disease. Interesting, endoplasmic reticulum (ER) stress is known to contribute to the pathophysiological mechanisms during the development of renal fibrosis. Here, we investigated the effects of chemical chaperon sodium 4-phenylbutyrate (4-PBA) on renal fibrosis in vivo and in vitro. In a rat unilateral ureteral obstruction (UUO) model, 4-PBA mimicked endogenous ER chaperon in the kidneys and significantly reduced glucose regulated protein 78 (GRP78), CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), activating transcription factor 4 (ATF4), and phosphorylated JNK protein expressions as well as restored spliced X-box-binding protein 1 (XBP1) expressions in the kidneys of UUO rats. 4-PBA also attenuated the increases of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) protein expressions, tubulointerstitial fibrosis, and apoptosis in the kidneys of UUO rats. Moreover, transforming growth factor (TGF)-β markedly increased ER stress-associated molecules, profibrotic factors, and apoptotic markers in the renal tubular cells (NRK-52E), all of which could be significantly counteracted by 4-PBA treatment. 4-PBA also diminished TGF-β-increased CTGF promoter activity and CTGF mRNA expression in NRK-52E cells. Taken together, our results indicated that 4-PBA acts as an ER chaperone to ameliorate ER stress-induced renal tubular cell apoptosis and renal fibrosis.
Project description:AB toxins such as ricin and cholera toxin (CT) consist of an enzymatic A domain and a receptor-binding B domain. After endocytosis of the surface-bound toxin, both ricin and CT are transported by vesicle carriers to the endoplasmic reticulum (ER). The A subunit then dissociates from its holotoxin, unfolds, and crosses the ER membrane to reach its cytosolic target. Since protein unfolding at physiological temperature and neutral pH allows the dissociated A chain to attain a translocation-competent state for export to the cytosol, the underlying regulatory mechanisms of toxin unfolding are of paramount biological interest. Here we report a biophysical analysis of the effects of anionic phospholipid membranes and two chemical chaperones, 4-phenylbutyric acid (PBA) and glycerol, on the thermal stabilities and the toxic potencies of ricin toxin A chain (RTA) and CT A1 chain (CTA1). Phospholipid vesicles that mimic the ER membrane dramatically decreased the thermal stability of RTA but not CTA1. PBA and glycerol both inhibited the thermal disordering of RTA, but only glycerol could reverse the destabilizing effect of anionic phospholipids. In contrast, PBA was able to increase the thermal stability of CTA1 in the presence of anionic phospholipids. PBA inhibits cellular intoxication by CT but not ricin, which is explained by its ability to stabilize CTA1 and its inability to reverse the destabilizing effect of membranes on RTA. Our data highlight the toxin-specific intracellular events underlying ER-to-cytosol translocation of the toxin A chain and identify a potential means to supplement the long-term stabilization of toxin vaccines.
Project description:Chronic graft-versus-host disease (cGVHD) is a marked complication of hematopoietic stem cell transplantation, and multiple organs can be affected by cGVHD-induced inflammation and fibrosis. In clinical settings, immunosuppressive agents have been the last resort to treat cGVHD. However, it has been only partially effective for cGVHD. Hence, efficacious treatment of cGVHD is eagerly awaited. Our previous work suggested that oxidative stress was elevated in cGVHD-disordered lacrimal glands and that epithelial-to-mesenchymal transition (EMT) was implicated in fibrosis caused by ocular cGVHD. In addition, our recent article demonstrated that thioredoxin interaction protein (TXNIP) and transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) were associated with the development of cGVHD. After our search for effective drugs, we chose tranilast to combat systemic cGVHD. Tranilast is known to (1) act as an inhibitor of the inflammatory molecules TXNIP and NF-?B and (2) exert anti-fibrotic, anti-EMT and anti-oxidative effects. To investigate the effectiveness of tranilast for cGVHD, we used an MHC-compatible, multiple minor histocompatibility antigen-mismatched murine model of cGVHD. Tranilast or a solvent-vehicle were orally given to the allogeneic bone marrow transplantation (allo-BMT) recipients from the day before allo-BMT (Day-1) to Day 27 after allo-BMT. Their cGVHD-vulnerable organs were collected Day 28 after allo-BMT and analyzed by using various methods such as histology, immunohistochemistry and immunoblotting. As indicated by our results, tranilast alleviated cGVHD-elicited inflammation and fibrosis by suppressing the expression and/or activation of TXNIP and NF-?B and preventing EMT. Taken together, although this strategy may not be a complete cure for cGVHD, tranilast could be a promising medication to ameliorate cGVHD-triggered disabling symptoms.
Project description:Mutations in the collagen genes COL4A1 and COL4A2 cause Mendelian eye, kidney and cerebrovascular disease including intracerebral haemorrhage (ICH), and common collagen IV variants are a risk factor for sporadic ICH. COL4A1 and COL4A2 mutations cause endoplasmic reticulum (ER) stress and basement membrane (BM) defects, and recent data suggest an association of ER stress with ICH due to a COL4A2 mutation. However, the potential of ER stress as a therapeutic target for the multi-systemic COL4A1 pathologies remains unclear. We performed a preventative oral treatment of Col4a1 mutant mice with the chemical chaperone phenyl butyric acid (PBA), which reduced adult ICH. Importantly, treatment of adult mice with the established disease also reduced ICH. However, PBA treatment did not alter eye and kidney defects, establishing tissue-specific outcomes of targeting Col4a1-derived ER stress, and therefore this treatment may not be applicable for patients with eye and renal disease. While PBA treatment reduced ER stress and increased collagen IV incorporation into BMs, the persistence of defects in BM structure and reduced ability of the BM to withstand mechanical stress indicate that PBA may be counter-indicative for pathologies caused by matrix defects. These data establish that treatment for COL4A1 disease requires a multipronged treatment approach that restores both ER homeostasis and matrix defects. Alleviating ER stress is a valid therapeutic target for preventing and treating established adult ICH, but collagen IV patients will require stratification based on their clinical presentation and mechanism of their mutations.