|Ahead of print publication
The convergence of fibrosis and immune pathways: 5-HT2 and 5-HT2B antagonists attenuate profibrotic phenotype in human pancreatic stellate cells by modulating signal transducer and activator of transcription 3 phosphorylation
Supriya Sharma1, Gaurav Pande2, Mohit Kumar Rai3, Durga Prasanna Misra3, Latika Gupta3, Vikas Agarwal3
1 Department of Surgical Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
2 Department of Medical Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
3 Department of Clinical Immunology and Rheumatology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
|Date of Submission||20-Jun-2020|
|Date of Acceptance||23-Jun-2020|
Department of Medical Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Immune mechanisms are believed to play an important role in the pathogenesis of pancreatic inflammation and fibrosis. Transforming growth factor β1-mediated mechanisms are well known; however, an effective treatment option is yet not available. Serotonin (5-HT) is known to induce fibrosis in carcinoid tumors and diseases such as scleroderma and blocking 5-HT has shown promise in the attenuation of fibrosis in animal models of scleroderma. Herein, we evaluated the role of 5-HT in pancreatic fibrosis and its reversal with antagonists of 5-HT. Methodology: Pancreatic stellate cells (PSCs) were cultured from the two patients of pancreatic cancer undergoing pancreaticoduodenectomy. PSCs were cultured in the presence of 5-HT and as per the disease mimicking strategy (PSCs cultured with 5-HT first followed by 5-HT antagonist (terguride) and 5-HT2Bantagonist (SB204741) for 24 h and pretreatment strategy, PSCs treated with 5-HT antagonists first followed by 5-HT. mRNA expression of profibrotic genes (col1A1, Col1A2, ACTA-2, and CTGF) and Western blot analysis of signal transducer and activator of transcription 3 (STAT-3) and Smad-3 proteins was carried out. Results: 5-HT-induced profibrotic genes mRNA expression in PSCs was significantly reversed by 5-HT antagonists, terguride and SB204741, to a large extent. Pretreatment strategy was more effective in the amelioration of profibrotic potential. SB204741, 5-HT2Bantagonist was more effective than terguride. 5-HT antagonists mediated their effect by reducing the phosphorylation of noncanonical pathway mediator pSTAT3 more significantly then canonical pathway mediator pSmad-3. Conclusion: 5-HT induces profibrotic potential in human PSCs and inhibitors of 5-HT receptors, Terguride and SB 204,741 ameliorate it by reducing the phosphorylation of STAT-3 and Smad-3, more so in pretreatment strategy. 5-HT inhibitors may have a potential to ameliorate fibrosis in pancreas.
Keywords: Pancreatic fibrosis, pancreatic stellate cells, SB204741, Smad-3, signal transducer and activator of transcription 3, terguride
|How to cite this URL:|
Sharma S, Pande G, Rai MK, Misra DP, Gupta L, Agarwal V. The convergence of fibrosis and immune pathways: 5-HT2 and 5-HT2B antagonists attenuate profibrotic phenotype in human pancreatic stellate cells by modulating signal transducer and activator of transcription 3 phosphorylation. Indian J Rheumatol [Epub ahead of print] [cited 2020 Oct 29]. Available from: https://www.indianjrheumatol.com/preprintarticle.asp?id=298043
| Introduction|| |
Immune events are the forerunners to fibrosis. Recent insights have lent fresh perspectives to the pathogenesis of fibrosis in various disorders, including chronic pancreatitis and pancreatic carcinoma. Insights from the pathogenetic mechanisms in prototype conventional fibrotic disorder such as systemic sclerosis may be evaluated in pancreatic fibrosis.
There have been considerable strides over the past decade in our understanding of the pathogenesis of pancreatic fibrosis since the original description of the technique of isolation of quiescent pancreatic stellate cells (PSCs) in rats and activated PSCs in humans. Fibrosis is a characteristic feature of not only chronic pancreatitis but also pancreatic carcinoma. In the activated state, PSCs are phenotypically close to myofibroblasts but remain distinguishable by the expression of neuroectodermal markers (not seen in fibroblasts) such as glial fibrillary acidic protein and neural cell adhesion molecule.
The link between serotonin (5-HT) and fibrosis is evident from the observations; carcinoid syndrome, that secrete lots of 5-HT, is characterized by tissue fibrosis affecting cardiac valves, lung, and skin,,, retroperitoneal fibrosis caused by ergot methysergide (5-HT2B antagonist) into methylergonovine (5-HT2B agonist) and increased expression of 5-HT1A/1B/5-HT2B receptors in the lungs of patients with idiopathic pulmonary fibrosis.,, Amongst rheumatic diseases, immunoglobulin G4-related disease, and primary Sjogren's syndrome are associated with autoimmune pancreatitis that lead to tumorous masses that may mimic pancreatic cancer. In a patient with autoimmune pancreatitis, elevated plasma and urinary levels of serotonin have been reported. Whether elevated plasma serotonin levels have any association with pancreatitis was not clear from this case report.
Upon pancreatic injury, 5-HT is released at the injured site and PSCs are activated, acquiring the ability to migrate and produce abundant amounts of extracellular matrix (ECM) proteins, such as collagen, fibronectin, and laminin in addition to their role in ECM turnover and inflammation. Cellular effects of 5-HT are mediated by seven different G-protein-coupled receptors class of receptors: 5-HT receptor subtypes (5-HTR1–HTR7). 5-HT Class 2 (5-HT2) receptors, especially subtypes 5-HT2A and 5-HT2B have been implicated in chronic fibrotic disorders such as retroperitoneal or pleural fibrosis, carcinoid heart disease, and liver fibrosis.,,
Antifibrotic potential of 5-HT receptor antagonists in human adult dermal fibroblasts derived from a scleroderma patient has been recently reported. 5-HT through its 5-HT2A receptors mediate the process of regeneration, whereas through 5-HT2B receptors may activate fibrosis. The role of 5-HT in mediating pancreatic fibrosis is not clear. In the present study, we evaluated the role of 5-HT in the induction of profibrotic genes mRNA expression and explored its potential for reversal with 5-HT receptor inhibitors.
| Methodology|| |
Culture of pancreatic stellate cells
A bloc of pancreatic tissue measuring 0.5/05 cm was obtained from the pancreatic neck or midbody during lateral pancreatojejunostomy for chronic pancreatitis in sterile saline. The tissues were incubated with phosphate-buffered saline containing 2% antibiotics for 15 min and then decanted. This step was repeated three times. PSCs were cultured from the pancreatic tissue blocks by the explant culture method.
Stimulation of pancreatic stellate cells
1.2 × 106 cells/well was seeded in six well plates for 24 h. Following adherence, cells were synchronized by incubating them with 2% Dulbecco's modified Eagle's medium (DMEM) for 24 h. Now, PSCs were incubated with 1 μM 5-HT. For the treatment with 5-HT2 and 5-HT2B inhibitors, two strategies were adopted. In thefirst posttreatment strategy (disease mimicking strategy), PSCs were initially treated with 5-HT (1 μM) for 1 h and later incubated with 5-HT (1 μM) and terguride or SB204741 (1 μM, each) for 24 h. In the second pretreatment strategy, cells were pretreated with terguride or SB204741 (1 μM, each) for 1 h and later stimulated with only 5-HT (1 μM) for 24 h. The cells were used for RNA and protein analysis.
Real-time polymerase chain reaction
Total RNA was extracted from PSCs using RNAiso Plus (Trizol method), and cDNA was prepared using cDNA synthesis kit as per the manufacturer's protocol. Quantification of mRNA by the real-time polymerase chain reaction (PCR) was carried out using Light Cycler® 480 real-time PCR system along with × 2 Maxima SYBR green RT-PCR master mix (Roche). The primers used are listed in [supplementary Table 1]. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard.
Gene expression analysis of human pluripotent stem cells
RNA was isolated from stimulated human pluripotent stem cells by Trizol method and real time PCR were performed for profibrotic gene expression (Col1a1, Col1a2, ASMA1, and CTGF) at mRNA level by sybergreen method. All the analysis was performed by using GAPDH as housekeeping gene.
Western blot studies
The cells were homogenized and sonicated in cell lysis buffer-containing protease and phosphatase inhibitors to extract whole cell lysate. Sample mixtures were then loaded onto 8%–10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels by the method of Laemmli. 30 μg of protein was used to perform the expression studies of phosphorylation status of Smad3 and signal transducer and activator of transcription 3 (STAT-3). The immunoreactive bands were visualized by enhanced chemiluminescence detection (GE Healthcare, Buckinghamshire, UK). The band intensity was measured by MYIMAGE analysis™ software (Thermo Scientific; Thermo Fisher Scientific, Waltham, MA, USA) using the spot densitometry analysis.
The study was approved by the Institutional Ethics Committee, and written informed consent was obtained from the patients as per the declaration of Helsinki.
All data are expressed as mean ± standard error of the mean for a minimum of three different experiments. All the groups were evaluated for the level of significance by using the Student's t-test. A probability value of ≤0.05 was considered as statistically significant.
| Results|| |
PSCs taken from the 3rd passage onward were stimulated at different doses with 5-HT (0.01 μM-10 μM). Viability of PSCs for 5-HT at 1 μM was 80%. 5-HT dose-dependently induced the profibrotic genes expression with maximal induction at a concentration of 1 μM (data not shown). Higher concentrations of 5-HT did not further increase the expression of pro-fibrotic genes mRNA.
Terguride and SB204741 attenuate profibrotic genes mRNA expression in pancreatic stellate cells
PSCs were incubated with terguride and SB204741 both at (0.01 μM-10 μM), respectively, along with 5-HT at 1 μM. Percentage viability for both the inhibitors at 1 μM was 80%. There was a decrease in mRNA levels of COL1A1, COL1A2, and ACTA2 genes in a dose-dependent manner (data not shown).
PSCs were synchronized with DMEM containing 2% fetal bovine serum for 24 h. Subsequently, cells were stimulated with 1 μM 5-HT, which up-regulated the levels of the COL1A1, COL1A2, ACTA2, and CTGF genes mRNA at 24 h [Figure 1]a, [Figure 1]b, [Figure 1]c, [Figure 1]d.
|Figure 1: Pancreatic stellate cells of two patients were cultured with media (bar 1) and with 5-HT (bar 2) and to mimic disease such as condition, Pancreatic stellate cells were firstly pre-stimulated with 5-HT (1 μ M) for 1 hour which is followed by co-culture with 5-HT (1 μ M) and 1 μ M dose of terguride (bar 3) and SB204741 (bar 4), respectively, for 24 hour. In the second strategy, pancreatic stellate cells were pre-treated with 1 μ M dose of terguride (bar 5) and SB204741 (bar 6), respectively, for 1 h and followed by incubation with 5-HT (1 μ M) for 24 h (a) Expression of mRNA levels of collagen type I alpha 1 chain, collagen type I alpha 2 chain, ACTA2, and connective tissue growth factor was normalized with glyceraldehyde 3-phosphate dehydrogenase as the housekeeping gene and mRNA fold change were calculated, and each experiment was performed three times. Data of all the experiments were shown as mean ± standard error of the mean. Student's t-test was statistical analysis and P value ≤ 0.05) considered as statistically significant. All the experiments were performed in triplicate|
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In posttreatment strategy (disease-mimicking conditions), PSCs were prestimulated with 1 μM of 5-HT for 1 h followed by 5-HT (1 μM) and terguride or SB204741 (1 μM, each) for 24 h. mRNA levels of COL1A1, COL1A2, ACTA2, and CTGF genes were significantly down-regulated by terguride and SB204741 [Figure 1]a, [Figure 1]b, [Figure 1]c, [Figure 1]d compared to the stimulation with 5-HT in PSCs.
In pretreatment strategy with both inhibitors, PSCs were pretreated with terguride or SB204741 (1 μM, each) for 1 h followed by 5-HT (1 μM) for 24 h. Levels of COL1A1, COL1A2, ACTA2, and CTGF genes mRNA were down regulated significantly as compared to 5-HT stimulated dermal fibroblasts [Figure 1]a, [Figure 1]b, [Figure 1]c, [Figure 1]d.
Terguride and SB204741 affect the phosphorylation of signal transducer and activator of transcription 3 and smad3
We next evaluated the effects of 5-HT2 and 5-HT2B antagonism on phosphorylation of smad3 (pSmad3) and phosphorylation of STAT3 (pSTAT3). Blockade of pSTAT3 can inhibit fibrosis in scleroderma skin fibroblasts. PSCs were incubated with 5-HT along with inhibitors of 5-HT2 and 5-HT2B. Cell lysates were collected for immunoblotting. 5-HT2 and 5-HT2B antagonism though inhibited phosphorylation of Smad3 and STAT3 [Figure 2]a. However, terguride and SB204741 reduced 5-HT induced pSTAT3 to a greater extent than pSmad3. [Figure 2]b. These results suggest that terguride and SB204741 mediate antifibrotic effect by predominantly inhibiting the pSTAT3.
|Figure 2: Pancreatic stellate cells of two patients were cultured with media (bar 1) and with 5-HT (bar 2) and to mimic disease like condition, Pancreatic stellate cells were firstly prestimulated with 5-HT (1 ƒÊ M) for 1 hour which is followed by co-culture with 5-HT (1 ƒÊ M) and 1 ƒÊ M dose of terguride (bar 3) and SB204741 (bar 4), respectively, for 24 h. In the second strategy, pancreatic stellate cells were pre-treated with 1 ƒÊ M dose of terguride (bar 5) and SB204741 (bar 6), respectively, for 1 h and followed by incubation with 5-HT (1 ƒÊ M) for 24 h (Expression of pSmad-3 (a) and expression of phosphorylation of signal transducer and activator of transcription 3 (b) were normalized with glyceraldehyde 3-phosphate dehydrogenase as the housekeeping protein fold change were calculated and each experiment was performed three times. Data of all the experiments were shown as mean ± standard error of the mean. Student's t-test was statistical analysis and P ≤ 0.05) considered as statistically significant. All the experiments were performed in triplicate|
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| Discussion|| |
PSCs are the major players of tissue repair and fibrosis in a setting of pancreatic injury/inflammatory damage. In the present study, 5-HT mediated profibrotic phenotype of PSCs is partially reversed by the treatment with 5-HT antagonists. Specific inhibitor of 5-HT2B receptor, SB204741, is able to reverse profibrotic potential of PSCs to a greater extent as compared to nonspecific inhibitor of 5-HT receptors, Terguride. In addition, pretreatment strategy with antagonists was much more effective in reducing the profibrotic potential of PSCs. 5-HT receptor inhibitors-mediated reversal of pro-fibrotic potential of PSCs by reducing the phosphorylation of STAT-3 and Smad-3.
There is enough evidence to suggest that PSCs play a crucial role in pancreatic fibrosis, especially in diseases such as chronic pancreatitis and pancreatic cancer. Once PSCs are activated, they transform into myofibroblast like phenotype and start producing lots of ECM proteins and may remain in a state of perpetual activation even in the absence of the initial trigger factors and keep contributing to fibrosis. Earlier, 5-HT has been reported to activate hepatic stellate cells and contribute to liver fibrosis and hepatocellular carcinoma.
5-HT may mediate its fibrotic action either directly through 5-HT2B receptor or by the conversion of latent transforming growth factor beta 1 (TGF-β1) in plasma to active TGF-β1, or by inducing the expression of TGF-β1, which may mediate fibrosis through Smad and nonSmad dependent pathways. It has been reported that 5-HT2B signaling leads to the activation of guanosine triphosphatase and extracellular regulated kinase pathway in fibroblasts. In our earlier study, we have reported that 5-HT2B receptor inhibitor, SB 204,741, not only ameliorates 5-HT induced pro-fibrotic potential, but also TGFβ1 induced pro-fibrotic potential of human adult dermal fibroblasts. In addition, this action of SB 204,741 is mediated by the decrease in STAT-3 phosphorylation. Similarly, SB 204,741-mediated reduction in profibrotic genes mRNA expression in PSCs by reducing the phosphorylation of STAT3 and Smad3 in the present study.
PSCs in the present study were isolated from pancreatic neck and midbody of the pancreas from two patients of pancreatic carcinoma undergoing pancreatoduodenectomy. In the present study, 5-HT2B receptor inhibitor reduces fibrotic potential of PSCs partially and may thus has potential to be evaluated for therapeutics in pancreatic carcinoma.
| Conclusion|| |
To conclude, 5-HT induce profibrotic potential in human PSCs and inhibitors of 5-HT receptors, Terguride and SB 204,741 ameliorate it by reducing the phosphorylation of STAT3 and Smad3, more so in pretreatment strategy. 5-HT inhibitors may have potential in cases of pancreatic fibrosis.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Gaddy JR, Vista ES, Robertson JM, Dedeke AB, Roberts VC, Klein WS, et al
. Rheumatic disease among Oklahoma tribal populations: A cross-sectional study. J Rheumatol 2012;39:1934-41.
Apte MV, Haber PS, Applegate TL, Norton ID, McCaughan GW, Korsten MA, et al.
Periacinar stellate shaped cells in rat pancreas – Identification, isolation, and culture. Gut 1998;43:128-33.
Bachem MG, Schneider E, Gross H, Weidenbach H, Schmid RM, Menke A, et al
. Identification, culture, and characterization of pancreatic stellate cells in rats and humans. Gastroenterology 1998;115:421-32.
Fries JF, Lindgren JA, Bull JM. Scleroderma-like lesions and the carcinoid syndrome. Arch Intern Med 1973;131:550-3.
Oates JA, Melmon K, Sjoerdsma A, Gillespie L, Mason DT. Release of a Kinin peptide in the carcinoid syndrome. Lancet 1964;1:514-7.
Pavlovic M, Saiag P, Lotz JP, Marinho E, Clerici T, Izrael V. Regression of sclerodermatous skin lesions in a patient with carcinoid syndrome treated by octreotide. Arch Dermatol 1995;131:1207-9.
Reimund E. Methysergide and retroperitoneal fibrosis. Lancet 1987;1:443.
Dumitrascu R, Kulcke C, Königshoff M, Kouri F, Yang X, Morrell N, et al
. Terguride ameliorates monocrotaline-induced pulmonary hypertension in rats. Eur Respir J 2011;37:1104-18.
Launay JM, Hervé P, Peoc'h K, Tournois C, Callebert J, Nebigil CG, et al
. Function of the serotonin 5-hydroxytryptamine 2B receptor in pulmonary hypertension. Nat Med 2002;8:1129-35.
Morecroft I, Heeley RP, Prentice HM, Kirk A, MacLean MR. 5-hydroxytryptamine receptors mediating contraction in human small muscular pulmonary arteries: Importance of the 5-HT1B receptor. Br J Pharmacol 1999;128:730-4.
Zhang L, Smyrk TC. Autoimmune pancreatitis and IgG4-related systemic diseases. Int J Clin Exp Pathol 2010;3:491-504.
Nobili E, Pezzilli R, Santini D, Campidelli C, Calculli L, Casadei R, et al
. Autoimmune pancreatitis associated with high levels of chromogranin a, serotonin and 5-hydroxyindoleacetic Acid. Case Rep Gastroenterol 2008;2:11-7.
Apte MV, Pirola RC, Wilson JS. Pancreatic stellate cells: A starring role in normal and diseased pancreas. Front Physiol 2012;3:344.
Grewal JS, Mukhin YV, Garnovskaya MN, Raymond JR, Greene EL. Serotonin 5-HT2A receptor induces TGF-β1 expression in mesangial cells via ERK: Proliferative and fibrotic signals. Am J Physiol Ren Physiol 1999;276:F922-30.
Königshoff M, Dumitrascu R, Udalov S, Amarie OV, Reiter R, Grimminger F, et al
. Increased expression of 5-hydroxytryptamine2A/B receptors in idiopathic pulmonary fibrosis: A rationale for therapeutic intervention. Thorax 2010;65:949-55.
Ruddell RG, Oakley F, Hussain Z, Yeung I, Bryan-Lluka LJ, Ramm GA, et al
. A role for serotonin (5-HT) in hepatic stellate cell function and liver fibrosis. Am J Pathol 2006;169:861-76.
Chaturvedi S, Misra DP, Prasad N, Rastogi K, Singh H, Rai MK, et al
. 5-HT2 and 5-HT2B antagonists attenuate pro-fibrotic phenotype in human adult dermal fibroblasts by blocking TGF-β1 induced non-canonical signaling pathways including STAT3 : Implications for fibrotic diseases like scleroderma. Int J Rheum Dis 2018;21:2128-38.
Sharma S, Pande G, Rai MK, Agarwal V. Indigenous primary culture protocols for human adult skin fibroblast, pancreatic stellate cells, and peritoneal fibroblasts. Indian J Rheumatol 2020; (in press).
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-5.
Apte M, Pirola R, Wilson J. The fibrosis of chronic pancreatitis: New insights into the role of pancreatic stellate cells. Antioxid Redox Signal 2011;15:2711-22.
Yang Q, Yan C, Yin C, Gong Z. Serotonin activated hepatic stellate cells contribute to sex disparity in hepatocellular carcinoma. Cell Mol Gastroenterol Hepatol 2017;3:484-99.
Dees C, Akhmetshina A, Zerr P, Reich N, Palumbo K, Horn A, et al
. Platelet-derived serotonin links vascular disease and tissue fibrosis. J Exp Med 2011;208:961-72.
El-Tanbouly DM, Wadie W, Sayed RH. Modulation of TGF-β/Smad and ERK signaling pathways mediates the anti-fibrotic effect of mirtazapine in mice. Toxicol Appl Pharmacol 2017;329:224-30.
Masson J, Emerit MB, Hamon M, Darmon M. Serotonergic signaling: Multiple effectors and pleiotropic effects. WIREs Membr Transp Signal 2012;1:685-713.
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