Article | Published:

Knockout of α-calcitonin gene-related peptide attenuates cholestatic liver injury by differentially regulating cellular senescence of hepatic stellate cells and cholangiocytes

Laboratory Investigation (2019) | Download Citation


α-Calcitonin gene-related peptide (α-CGRP) is a 37-amino acid neuropeptide involved in several pathophysiological processes. α-CGRP is involved in the regulation of cholangiocyte proliferation during cholestasis. In this study, we aimed to evaluate if α-CGRP regulates bile duct ligation (BDL)-induced liver fibrosis by using a α-CGRP knockout (α-CGRP-/-) mouse model. α-CGRP-/- and wild-type (WT) mice were subjected to sham surgery or BDL for 7 days. Then, liver fibrosis and cellular senescence as well as the expression of kinase such as p38 and C-Jun N-terminal protein kinase (JNK) in mitogen-activated protein kinases (MAPK) signaling pathway were evaluated in total liver, together with measurement of cellular senescence in cholangiocytes or hepatic stellate cells (HSCs). There was enhanced hepatic expression of Calca (coding α-CGRP) and the CGRP receptor components (CRLR, RAMP-1 and RCP) in BDL and in both WT α-CGRP-/- and BDL α-CGRP-/- mice, respectively. Moreover, there was increased CGRP serum levels and hepatic mRNA expression of CALCA and CGRP receptor components in late-stage PSC samples compared to healthy control samples. Depletion of α-CGRP reduced liver injury and fibrosis in BDL mice that was associated with enhanced cellular senescence of hepatic stellate cells and reduced senescence of cholangiocytes as well as decreased activation of p38 and JNK MAPK signaling pathway. Cholangiocyte supernatant from BDL α-CGRP-/- mice inhibited the activation and increased cellular senescence of cultured human HSCs (HHSCs) compared to HHSCs stimulated with BDL cholangiocyte supernatant. Taken together, endogenous α-CGRP promoted BDL-induced cholestatic liver fibrosis through differential changes in senescence of HSCs and cholangiocytes and activation of p38 and JNK signaling. Modulation of α-CGRP/CGRP receptor signaling may be key for the management of biliary senescence and liver fibrosis in cholangiopathies.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Present address for Drs. Bernuzzi and Invernizzi: Program for Autoimmune Liver Diseases, International Center for Digestive Health, Department of Medicine and Surgery, University of Milan-Bicocca, Milano, Italy.


  1. 1.

    Mogler C, Wieland M, König C, Hu J, Runge A, Korn C, et al. Hepatic stellate cell-expressed endosialin balances fibrogenesis and hepatocyte proliferation during liver damage. EMBO Mol Med. 2015;7:332–8.

  2. 2.

    Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology. 2008;134:1655–69.

  3. 3.

    Rahimi RS, Rockey DC. Complications of cirrhosis. Curr Opin Gastroenterol. 2012;28:223–9.

  4. 4.

    Poynard T, Lebray P, Ingiliz P, Varaut A, Varsat B, Ngo Y, et al. Prevalence of liver fibrosis and risk factors in a general population using non-invasive biomarkers (FibroTest). BMC Gastroenterol. 2010;10:40.

  5. 5.

    Hernandez-Gea V, Friedman SL. Pathogenesis of liver fibrosis. Annu Rev Pathol. 2011;6:425–56.

  6. 6.

    Krizhanovsky V, Yon M, Dickins RA, Hearn S, Simon J, Miething C, et al. Senescence of activated stellate cells limits liver fibrosis. Cell. 2008;134:657–67.

  7. 7.

    Zhang X, Han X, Yin L, Xu L, Qi Y, Xu Y, et al. Potent effects of dioscin against liver fibrosis. Sci Rep. 2015;5:9713.

  8. 8.

    Tabibian JH, Trussoni CE, O’Hara SP, Splinter PL, Heimbach JK, LaRusso NF. Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis. Lab Invest. 2014;94:1126–33.

  9. 9.

    Baghdasaryan A, Claudel T, Gumhold J, Silbert D, Adorini L, Roda A, et al. Dual farnesoid X receptor/TGR5 agonist INT-767 reduces liver injury in the Mdr2-/- (Abcb4-/-) mouse cholangiopathy model by promoting biliary HCO(-)(3) output. Hepatology. 2011;54:1303–12.

  10. 10.

    Wan Y, Meng F, Wu N, Zhou T, Venter J, Francis H, et al. Substance P increases liver fibrosis by differential changes in senescence of cholangiocytes and hepatic stellate cells. Hepatology. 2017;66:528–41.

  11. 11.

    Tabibian JH, O'Hara SP, Splinter PL, Trussoni CE, LaRusso NF. Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis. Hepatology. 2014;59:2263–75.

  12. 12.

    Meng L, Quezada M, Levine P, Han Y, McDaniel K, Zhou T, et al. Functional role of cellular senescence in biliary injury. Am J Pathol. 2015;185:602–9.

  13. 13.

    Holzer P. Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience. 1988;24:739–68.

  14. 14.

    Barry CM, Kestell G, Gillan M, Haberberger RV, Gibbins IL. Sensory nerve fibers containing calcitonin gene-related peptide in gastrocnemius, latissimus dorsi and erector spinae muscles and thoracolumbar fascia in mice. Neuroscience. 2015;291:106–17.

  15. 15.

    Glaser SS, Ueno Y, DeMorrow S, Chiasson VL, Katki KA, Venter J, et al. Knockout of alpha-calcitonin gene-related peptide reduces cholangiocyte proliferation in bile duct ligated mice. Lab Invest. 2007;87:914–26.

  16. 16.

    Henriksen JH, Schifter S, Møller S, Bendtsen F. Increased circulating calcitonin in cirrhosis. Relation to severity of disease and calcitonin gene-related peptide. Metabolism. 2000;49:47–52.

  17. 17.

    Yoon SP, Kim J Exogenous CGRP upregulates profibrogenic growth factors through PKC/JNK signaling pathway in kidney proximal tubular cells. Cell Biol Toxicol 2017;4:251–62.

  18. 18.

    Glaser S, Benedetti A, Marucci L, Alvaro D, Baiocchi L, Kanno N, et al. Gastrin inhibits cholangiocyte growth in bile duct-ligated rats by interaction with cholecystokinin-B/Gastrin receptors via D-myo-inositol 1,4,5-triphosphate-, Ca(2+)-, and protein kinase C alpha-dependent mechanisms. Hepatology. 2000;32:17–25.

  19. 19.

    Puche JE, Lee YA, Jiao J, Aloman C, Fiel MI, et al. A novel murine model to deplete hepatic stellate cells uncovers their role in amplifying liver damage in mice. Hepatology. 2013;57:339–50.

  20. 20.

    Schmitt-Gräff A, Krüger S, Bochard F, Gabbiani G, Denk H. Modulation of alpha smooth muscle actin and desmin expression in perisinusoidal cells of normal and diseased human livers. Am J Pathol. 1991;138:1233–42.

  21. 21.

    Zhang L, Hoff AO, Wimalawansa SJ, Cote GJ, Gagel RF, Westlund KN. Arthritic calcitonin/alpha calcitonin gene-related peptide knockout mice have reduced nociceptive hypersensitivity. Pain. 2001;89:265–73.

  22. 22.

    Glaser S, Gaudio E, Renzi A, Mancinelli R, Ueno Y, Venter J. Knockout of the neurokinin-1 receptor reduces cholangiocyte proliferation in bile duct-ligated mice. Am J Physiol Gastrointest Liver Physiol. 2011;301:G297–305.

  23. 23.

    Glaser S, Meng F, Han Y, Onori P, Chow BK, Francis H, et al. Secretin stimulates biliary cell proliferation by regulating expression of microRNA 125b and microRNA let7a in mice. Gastroenterology. 2014;146:1795–808 e1712.

  24. 24.

    Wu N, Meng F, Invernizzi P, Bernuzzi F, Venter J, Standeford H, et al. The secretin/secretin receptor axis modulates liver fibrosis through changes in transforming growth factor-beta1 biliary secretion in mice. Hepatology. 2016;64:865–79.

  25. 25.

    Ballardini G, Fallani M, Biagini G, Bianchi FB, Pisi E. Desmin and actin in the identification of Ito cells and in monitoring their evolution to myofibroblasts in experimental liver fibrosis. Virchows Arch B Cell Pathol Incl Mol Pathol. 1988;56:45–49.

  26. 26.

    Ehrlich L, O'Brien A, Hall C, White T, Chen L, Wu N, et al. alpha7-nAChR Knockout Mice Decreases Biliary Hyperplasia and Liver Fibrosis in Cholestatic Bile Duct-Ligated Mice. Gene Expr. 2018;18:197–207.

  27. 27.

    Bansal R, Prakash J, De Ruiter M, Poelstra K. Interferon gamma peptidomimetic targeted to hepatic stellate cells ameliorates acute and chronic liver fibrosis in vivo. J Control Release. 2014;179:18–24.

  28. 28.

    Mitra A, Satelli A, Yan J, Xueqing X, Gagea M, Hunter CA, et al. IL-30 (IL27p28) attenuates liver fibrosis through inducing NKG2D-rae1 interaction between NKT and activated hepatic stellate cells in mice. Hepatology. 2014;60:2027–39.

  29. 29.

    Li WW, Guo TZ, Shi X, Birklein F, Schlereth T, Kingery WS, et al. Neuropeptide regulation of adaptive immunity in the tibia fracture model of complex regional pain syndrome. J Neuroinflammation. 2018;15:105.

  30. 30.

    Sheykhzade M, Abdolalizadeh B, Koole C, Pickering DS, Dreisig K, Johansson SE, et al. Vascular and molecular pharmacology of the metabolically stable CGRP analogue, SAX. Eur J Pharmacol. 2018;829:85–92.

  31. 31.

    Franco-Cereceda A, Lundberg JM. Calcitonin gene-related peptide (CGRP) and capsaicin-induced stimulation of heart contractile rate and force. Naunyn Schmiedebergs Arch Pharmacol. 1985;331:146–51.

  32. 32.

    Andersen SL, Clausen T. Calcitonin gene-related peptide stimulates active Na(+)-K+ transport in rat soleus muscle. Am J Physiol Cell Physiol. 1993;264:C419–429. (2 Pt 1)

  33. 33.

    Li Y, Fiscus RR, Wu J, Yang L, Wang X. The antiproliferative effects of calcitonin gene-related peptide in different passages of cultured vascular smooth muscle cells. Neuropeptides. 1997;31:503–9.

  34. 34.

    Tache Y. Inhibition of gastric acid secretion and ulcers by calcitonin [correction of calciton] gene-related peptide. Ann N Y Acad Sci. 1992;657:240–7.

  35. 35.

    Kim J, Padanilam BJ. Renal nerves drive interstitial fibrogenesis in obstructive nephropathy. J Am Soc Nephrol. 2013;24:229–42.

  36. 36.

    Sasaki M, Ikeda H, Yamaguchi J, Miyakoshi M, Sato Y, Nakanuma Y. Bile ductular cells undergoing cellular senescence increase in chronic liver diseases along with fibrous progression. Am J Clin Pathol. 2010;133:212–23.

  37. 37.

    Beuers U. Crosstalk of liver, bile ducts and the gut. Clin Rev Allergy Immunol. 2009;36:1–3.

  38. 38.

    Li XW, Li XH, Du J, Li D, Li YJ, Hu CP. Calcitonin gene-related peptide down-regulates bleomycin-induced pulmonary fibrosis. Can J Physiol Pharmacol. 2016;94:1315–24.

  39. 39.

    Kamiyoshi A, Sakurai T, Ichikawa-Shindo Y, Iinuma N, Kawate H, Yoshizawa T, et al. Endogenous alpha-calcitonin gene-related peptide mitigates liver fibrosis in chronic hepatitis induced by repeated administration of concanavalin A. Liver Int. 2009;29:642–9.

Download references


This work was supported by the Dr. Nicholas C. Hightower Centennial Chair of Gastroenterology from Baylor Scott & White, a VA Research Career Scientist Award and a VA Merit award to Dr. Alpini (5I01BX000574), a VA Merit Award (1I01BX001724) to Dr. Meng, and the NIH grants DK058411, DK076898, DK107310, DK110035, DK062975, AA025997, and AA025157 to Drs. Alpini, Meng, and Glaser. Portions of this work were supported by (i) a VA Merit Award (1I01BX003031) from the United States Department of Veteran’s affairs, Biomedical Laboratory Research and Development Service and an R01 from NIH NIDDK (DK108959) to Dr. Francis (ii) and a Nature Science foundation of China (No.81873563) to Dr. Wan. This material is the result of work supported with resources and the use of facilities at the Central Texas Veterans Health Care System, Temple, Texas.

Author information

Author notes

  1. Ying Wan and Ludovica Ceci share the first authorship.

  2. Drs. Gianfranco Alpini, Fanyin Meng, and Shannon Glaser share the senior authorship.


  1. Department of Pathophysiology, Southwest Medical University, Luzhou, 646000, China

    • Ying Wan
  2. Department of Medical Physiology, Texas A&M University Health Science Center, Temple, TX, USA

    • Ludovica Ceci
    • , Nan Wu
    • , Tianhao Zhou
    • , Lixian Chen
    • , Julie Venter
    • , Heather Francis
    • , Konstantina Kyritsi
    • , Paul Baker
    • , Gianfranco Alpini
    • , Fanyin Meng
    •  & Shannon Glaser
  3. Research, Central Texas Veterans Health Care System, Temple, TX, USA

    • Heather Francis
    • , Gianfranco Alpini
    •  & Fanyin Meng
  4. Digestive Disease Research Center, Baylor Scott & White Health, Temple, TX, USA

    • Heather Francis
    • , Gianfranco Alpini
    • , Fanyin Meng
    •  & Shannon Glaser
  5. Humanitas Clinical and Research Center, Rozzano (MI), Italy

    • Francesca Bernuzzi
    •  & Pietro Invernizzi
  6. Department of Pathophysiology, Key Lab for Shock and Microcirculation Research of Guangdong Province, Southern Medical University, Guangzhou, China

    • Qiaobing Huang
  7. Department of Nutrition and Food Science, Texas A&M University, College Station, TX, 77843, USA

    • Chaodong Wu
  8. Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA

    • Amelia Sybenga
  9. Research Foundation, Baylor Scott & White Health, Temple, TX, USA

    • Fanyin Meng


  1. Search for Ying Wan in:

  2. Search for Ludovica Ceci in:

  3. Search for Nan Wu in:

  4. Search for Tianhao Zhou in:

  5. Search for Lixian Chen in:

  6. Search for Julie Venter in:

  7. Search for Heather Francis in:

  8. Search for Francesca Bernuzzi in:

  9. Search for Pietro Invernizzi in:

  10. Search for Konstantina Kyritsi in:

  11. Search for Paul Baker in:

  12. Search for Qiaobing Huang in:

  13. Search for Chaodong Wu in:

  14. Search for Amelia Sybenga in:

  15. Search for Gianfranco Alpini in:

  16. Search for Fanyin Meng in:

  17. Search for Shannon Glaser in:

Conflict of interest

The authors declare that they have no conflict of interest.

Corresponding author

Correspondence to Gianfranco Alpini.

Supplementary information

About this article

Publication history