Gastroenterology
Volume 135, Issue 2 , Pages 347-351, August 2008

Hedgehog Spikes Pancreas Regeneration

  • David A. Cano

      Affiliations

    • Instituto de Biomedicina de Sevilla, Servicio de Endocrinología y Nutrición, Hospitales Universitarios Virgen del Rocío, Sevilla, Spain
    • ,
  • Matthias Hebrok

      Affiliations

    • Diabetes Center, Department of Medicine, University of California, San Francisco, California
    • Corresponding Author InformationAddress requests for reprints to: Matthias Hebrok, Diabetes Center, Department of Medicine, University of California, San Francisco, CA 94143; Fax: (415) 564-5813.

published online 11 July 2008.

Article Outline

 

See “Hedgehog signaling is required for effective regeneration of exocrine pancreas,” by Fendrich V, Esni F, Garay MV, et al, on page 621.

The adult pancreas remains a mysterious organ. Not only does it form from distinct epithelial buds derived from both the dorsal and the ventral gut epithelium, it also harbors diverse cell types with dramatically different functions (reviewed in Cano et al1). The exocrine compartment is composed of acinar cells that produce and secrete digestive enzymes into a branched ductal system that drains into the intestine. The endocrine islets of Langerhans produce hormones that are released directly into the blood stream to regulate glucose homeostasis. Given the disparate morphologic and functional properties, it is noteworthy that all the mature pancreatic cell types originate from the same common embryonic progenitors during pancreatic development.2 Although significant progress has been made over the last few years toward the identification of the embryonic pancreatic progenitor cells and the transcriptional signaling cascades that regulate their stem cell–like state, little information is known about the mechanisms that underlie regeneration in adult pancreatic cells.

The adult pancreas is perceived commonly as a tissue with low cell turnover and restricted regeneration potential.3 However, diverse forms of tissue injury, including partial pancreatoctomy and pancreatic duct ligation in animal models, demonstrate an unexpected regenerative ability.4, 5, 6, 7, 8 More specific elimination of distinct cell types has revealed that both endocrine and exocrine cells possess the potential to restore their cell numbers upon insult. For example, efficient regeneration of beta cells is achieved within 2–3 months after almost complete ablation of beta cells in transgenic mice.9, 10 Similarly, destruction of acinar cells via treatment with cerulein, an agonist of the gastrointestinal hormone cholecystokinin that regulates secretion of exocrine enzymes, reveals a remarkable regenerative ability of these cells.11, 12 Cerulein treatment in rodents has been traditionally used as a model of human acute and chronic pancreatitis (reviewed in Saluja et al13). Administration of the agent at supramaximal levels results in pancreatic edema and an almost complete loss of acinar cells. However, despite the severe damage, full recovery of the acinar cell compartment is achieved within 1 week and detailed analysis of cerulein-induced pancreatitis has proven to be a useful tool to investigate the mechanisms underlying exocrine pancreatic regeneration. These studies have revealed that cerulein treatment induces acinar-to-ductal metaplasia, a process in which acinar cells are replaced by duct-like epithelium.11, 12 More recently, lineage tracing experiments tagging the mature acinar cells via an irreplaceable genetic marker indicate that acinar-to-ductal metaplasia following cerulein treatment involves the transdifferentiation of acinar cells toward a transient metaplastic epithelium.14 After cessation of the insult, the metaplastic epithelium differentiates back toward the acinar phenotype. These data suggest that during pancreatitis acinar cells transiently acquire ductal properties and that these cells redifferentiate into acinar cells. This conclusion is sustained by results from studies in which exocrine regeneration was induced by partial pancreatoctomy,15 supporting the notion that there is no significant contribution of other cell types during acinar regeneration.

Molecular evidence has been gathered to complement the morphologic data of exocrine regeneration. Cerulein-mediated injury and posterior regeneration seems to activate the expression of genes expressed typically during embryonic pancreatic development such as pancreatic and duodenal homeobox gene 1 (Pdx1) or clusterin.12 Reactivation of Notch signaling, a pathway that plays an important role in pancreas organogenesis,16, 17, 18 has been observed also during regeneration.12, 19 Support for a functional requirement of Notch signaling comes from studies in which chemical and genetic inactivation of Notch signaling in mice leads to impaired regeneration of the exocrine pancreas.19 In summary, these results indicate that exocrine regeneration after cerulein-induced injury recapitulates several aspects of normal embryonic development.

In this issue of Gastroenterology, Fendrich et al20 further strengthen this notion by reporting that Hedgehog (Hh) signaling, another embryonic signaling pathway, is required for regeneration of the exocrine pancreas in mice. Following a protocol for cerulein treatment shown previously to result in almost complete loss of exocrine cells,12 the authors report rapid activation of Hh signaling components during exocrine injury and regeneration. In normal adult pancreas, Hh signaling activity seems to be very low. However, expression of ligands (Shh and Ihh) and Hh-target genes (Ptch1 and Gli1) is observed almost immediately after the last cerulein injection. After regeneration, the expression of Hh components returns to basal levels, suggesting that the transient increase in Hh signaling might be necessary for certain aspects of the regeneration process. To test this hypothesis, the authors decided to inactivate Hh signaling by chemical and genetic means. Cyclopamine, a steroidal alkaloid that binds to an essential component of the Hh pathway, Smoothened, was administered to mice to inhibit the pathway. As expected, cyclopamine treatment blocked the increase in Hh activity upon cerulein-induced regeneration. Remarkably, Hh inhibition efficiently impaired the exocrine regeneration response, indicating a crucial role for Hh signaling in this process. Further proof for this hypothesis came from genetic experiments in which the Cre/lox technology was used to conditionally inactivate Smoothened in pancreatic cells. Inactivation in epithelial cells was accomplished using a transgenic mouse expressing the Cre recombinase under the control of the Pdx1 promoter, a gene that is expressed in all pancreatic cells at some point during development (Pdx-Cre;Smoflox/flox). In addition, Smoothened was inactivated more specifically in acinar cells when the expression of a tamoxifen-inducible Cre was regulated by the Elastase promoter (Ela-CreERT2;Smoflox/flox). As observed before in the cyclopamine/cerulein-treated mice, Hh inhibition in Smo-negative mice resulted in impaired regenerative response upon cerulein injection. Further analysis revealed that similar to controls, mice deficient in Hh activity develop metaplastic lesions after cerulein-induced injury. However, in contrast to the normal pancreatic tissue, exocrine cells with impaired Hh signaling have lost their ability for full regeneration after the metaplastic stage. Thus, Hh signaling seems to be necessary only for the differentiation process of the undifferentiated metaplastic epithelium into mature acinar cells (Figure 1). Interestingly, Pdx-Cre;Smoflox/flox mice do not show any obvious exocrine abnormalities during embryonic development or organ maturation, indicating that the role of Hh signaling during cerulein-induced acinar differentiation appears to be specific to adult tissue.

  • View full-size image.
  • Figure 1. 

    Inactivation of Hedgehog signaling impairs acinar cell regeneration. Pancreatic injury induced by caerulein administration leads to acinar cell loss and formation of a metaplastic epithelium. This metaplastic epithelium is characterized by the re-activation of genes typically expressed in the developing pancreas, including Pdx1 and Nestin. Wild type acinar cells regenerate completely upon cessation of the insult. In contrast, acinar cells marked by loss of Hedgehog signaling are impaired in their regenerative abilities.

Regenerative processes after organ injury are essential for tissue homeostasis and include the activation and proliferation of residing adult stem/progenitor cells. Alternatively, the process can be achieved by activating the replicative process in a facultative progenitor cell, that is, a mature cell type that can acquire traits of progenitor cells in the face of injury. Such facultative progenitor cells are commonly found in slowly renewing tissues, such as the liver (hepatocyte) and thymus (cortical and medullary epithelial cells).21, 22 The Hh signaling pathway has previously been implicated in tissue homeostasis and regeneration in other organs.23, 24, 25, 26, 27 In these studies, Hh activity has been reported to control expansion of the few adult stem/progenitor cells present in the tissues under normal conditions. It has also been suggested that Hh activity might be involved in activation of facultative progenitor cells in adult liver injury, but no functional data have been presented to support this hypothesis.28 The results described by Fendrich et al20 report a requirement of Hh signaling for activation of facultative progenitor cells during acinar cell regeneration, thus adding a novel role for the Hh pathway. The rapid activation of the Hh signaling pathway in acinar cells upon cerulein-mediated injury is astounding, particularly considering that no Hh activity is found in mature acinar cells under normal conditions. A possible explanation for the rapid elevation of Hh activity might lie in the observation that acinar cells produce both Hh ligands and the Hh receptor Patched (Ptch), suggesting the presence of an autocrine loop in acinar cells during regeneration. The absence of Hh signaling in normal acinar cells also raises questions about the nature of the triggering event that promotes the induction of Hh ligand expression during cerulein-induced injury. Injury usually results in the release of proinflammatory cytokines and growth factors by damaged cells or inflammatory cells. Some of these signals could be responsible for Shh activation in cerulein-induced injury. In this context, it is important to note that the growth factors Tgfβ1 and platelet-derived growth factor-BB have been shown to induce Hh ligands production in other tissues, including the liver.29, 30 In addition, nuclear factor (NF)-κB seems to play a central role in the inflammatory response induced by cerulein.31 Recent studies have described NF-κB activation as one of the mechanisms underlying Shh overexpression in pancreatic cancer.32 Given that pancreatitis is a strong risk factor for pancreatic cancer (discussed below), it would be interesting to determine whether NF-κB activation is involved in Hh-mediated exocrine pancreatic regeneration and whether common mechanisms regulate aspects in both scenarios.

The genetic experiments described by Fendrich et al20 suggest that specific activation of Hh in acinar cells is necessary for the regenerative response. These results are in agreement with lineage tracing experiments confirming the role of preexisting acinar cells as the origin of newly formed acinar cells during regeneration.14, 15 However, as the authors acknowledge, their data cannot completely rule out the role of a progenitor cell in this process. In this context, it is important to note that the existence of an uncommitted progenitor cell capable of promoting endocrine cell differentiation has recently been indicated by Heimberg et al.4 It would be interesting to determine whether these recently identified adult progenitor cells located in ducts could also play a role in exocrine regeneration in the cerulein-induced injury model.

Additional questions remain about the nature of the transient, undifferentiated metaplastic epithelium that develops after cerulein injury (and is maintained in Hh-deficient mice). In agreement with prior reports, some by the same authors, Fendrich et al20 describe the up-regulation of embryonic markers, including nestin, a gene expressed during embryonic acinar cell formation, in regenerating metaplastic epithelium. Based on morphologic features and cell of origin, the metaplastic epithelium seems to correspond to the type 1 tubular complexes observed in the setting of chronic pancreatitis recently described by Sarah Thayer's group.14 Interestingly, activation of embryonic pathways (Hh, Notch) has also been observed in human pancreatitis,32, 33, 34 suggesting a possible attempt of exocrine cells to regenerate in the human organ. These results suggest that injury can cause a dedifferentiation process in acinar cells leading to an immature state. Although similar, dedifferentiated cells of the metaplastic epithelium do not regress to a state in which they recapitulate the cell phenotype of fully uncommitted embryonic progenitor cells.12 Furthermore, under the conditions tested, the acinar cell-derived metaplastic epithelium does not possess the ability to generate endocrine or ductal cells (Fendrich et al20). Additional characterization of the metaplastic epithelium will be necessary to determine the exact nature of the dedifferentiated cells.

Another important aspect of the findings reported by Fendrich et al20 is the possible relevance of pancreatic epithelial plasticity as a mechanism underlying pancreatic tumor formation. Patients with ductal metaplasia arising in the setting of chronic pancreatitis have a 16-fold increase in relative risk for pancreatic ductal adenocarcinoma.35, 36 An increasing body of evidence indicates that uncontrolled activation of the Hh pathway is a critical step during the formation of pancreatic adenocarcinoma.37, 38 The results by Fendrich et al20 might suggest that activation of Hh is one link between chronic inflammation of the pancreas and carcinogenesis. To this regard, it should be noted that cerulein-induced pancreatitis in the context of a k-Ras mutation causes extensive formation of PanIN lesions, common precursors of pancreatic adenocarcinoma.39 It would be interesting to determine whether Hh is up-regulated in this model and whether inhibition of Hh signaling would result in blocking of cerulein-induced PanIN formation.

In summary, the report by Fendrich et al20 provides provocative data linking the Hh signaling pathway to pancreas acinar regeneration. Future studies need to address whether the positive effect of the pathway during normal regeneration can be high jacked in diseased pancreas marked by the expression of k-Ras mutation that sensitize toward pancreatic adenocarcinoma. Under these conditions, inhibition, rather than stimulation of Hh signaling, could prevent unwanted proliferative responses resulting in pancreatic cancer.

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References 

  1. Cano DA, Hebrok M, Zenker M. Pancreatic development and disease. Gastroenterology. 2007;132:745–762
  2. Gu G, Dubauskaite J, Melton DA. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development. 2002;129:2447–2457
  3. Teta M, Long SY, Wartschow LM, et al. Very slow turnover of beta-cells in aged adult mice. Diabetes. 2005;54:2557–2567
  4. Xu X, D'Hoker J, Stange G, et al. Beta cells can be generated from endogenous progenitors in injured adult mouse pancreas. Cell. 2008;132:197–207
  5. Teta M, Rankin MM, Long SY, et al. Growth and regeneration of adult beta cells does not involve specialized progenitors. Dev Cell. 2007;12:817–826
  6. Dor Y, Brown J, Martinez OI, et al. Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature. 2004;429:41–46
  7. Bonner-Weir S, Weir GC. New sources of pancreatic beta-cells. Nat Biotechnol. 2005;23:857–861
  8. Bonner-Weir S, Baxter LA, Schuppin GT, et al. A second pathway for regeneration of adult exocrine and endocrine pancreas (A possible recapitulation of embryonic development). Diabetes. 1993;42:1715–1720
  9. Cano DA, Rulifson IC, Heiser PW, et al. Regulated beta-cell regeneration in the adult mouse pancreas. Diabetes. 2008;57:958–966
  10. Nir T, Melton DA, Dor Y. Recovery from diabetes in mice by beta cell regeneration. J Clin Invest. 2007;117:2553–2561
  11. Reid LE, Walker NI. Acinar cell apoptosis and the origin of tubular complexes in caerulein-induced pancreatitis. Int J Exp Pathol. 1999;80:205–215
  12. Jensen JN, Cameron E, Garay MV, et al. Recapitulation of elements of embryonic development in adult mouse pancreatic regeneration. Gastroenterology. 2005;128:728–741
  13. Saluja AK, Lerch MM, Phillips PA, et al. Why does pancreatic overstimulation cause pancreatitis?. Annu Rev Physiol. 2007;69:249–269
  14. Strobel O, Dor Y, Alsina J, et al. In vivo lineage tracing defines the role of acinar-to-ductal transdifferentiation in inflammatory ductal metaplasia. Gastroenterology. 2007;133:1999–2009
  15. Desai BM, Oliver-Krasinski J, De Leon DD, et al. Preexisting pancreatic acinar cells contribute to acinar cell, but not islet beta cell, regeneration. J Clin Invest. 2007;117:971–977
  16. Jensen J, Pedersen EE, Galante P, et al. Control of endodermal endocrine development by Hes-1. Nat Genet. 2000;24:36–44
  17. Apelqvist A, Li H, Sommer L, et al. Notch signalling controls pancreatic cell differentiation. Nature. 1999;400:877–881
  18. Esni F, Ghosh B, Biankin AV, et al. Notch inhibits Ptf1 function and acinar cell differentiation in developing mouse and zebrafish pancreas. Development. 2004;131:4213–4224
  19. Siveke JT, Lubeseder-Martellato C, Lee M, et al. Notch signaling is required for exocrine regeneration after acute pancreatitis. Gastroenterology. 2008;134:544–555
  20. Fendrich V, Esni F, Garay MV, et al. Hedgehog signaling is required for effective regeneration of exocrine pancreas. Gastroenterology. 2008;135:621–631
  21. Bennett AR, Farley A, Blair NF, et al. Identification and characterization of thymic epithelial progenitor cells. Immunity. 2002;16:803–814
  22. Blau HM, Brazelton TR, Weimann JM. The evolving concept of a stem cell: entity or function?. Cell. 2001;105:829–841
  23. Beachy PA, Karhadkar SS, Berman DM. Tissue repair and stem cell renewal in carcinogenesis. Nature. 2004;432:324–331
  24. Machold R, Hayashi S, Rutlin M, et al. Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches. Neuron. 2003;39:937–950
  25. Watkins DN, Berman DM, Burkholder SG, et al. Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer. Nature. 2003;422:313–317
  26. Karhadkar SS, Bova GS, Abdallah N, et al. Hedgehog signalling in prostate regeneration, neoplasia and metastasis. Nature. 2004;431:707–712
  27. Trowbridge JJ, Scott MP, Bhatia M. Hedgehog modulates cell cycle regulators in stem cells to control hematopoietic regeneration. Proc Natl Acad Sci U S A. 2006;103:14134–14139
  28. Omenetti A, Diehl AM. The adventures of sonic hedgehog in development and repair (II. Sonic hedgehog and liver development, inflammation, and cancer). Am J Physiol Gastrointest Liver Physiol. 2008;294:G595–G598
  29. Yang L, Wang Y, Mao H, et al. Sonic hedgehog is an autocrine viability factor for myofibroblastic hepatic stellate cells. J Hepatol. 2008;48:98–106
  30. Omenetti A, Yang L, Li YX, et al. Hedgehog-mediated mesenchymal-epithelial interactions modulate hepatic response to bile duct ligation. Lab Invest. 2007;87:499–514
  31. Altavilla D, Famulari C, Passaniti M, et al. Attenuated cerulein-induced pancreatitis in nuclear factor-kappaB-deficient mice. Lab Invest. 2003;83:1723–1732
  32. Nakashima H, Nakamura M, Yamaguchi H, et al. Nuclear factor-kappaB contributes to hedgehog signaling pathway activation through sonic hedgehog induction in pancreatic cancer. Cancer Res. 2006;66:7041–7049
  33. Bhanot U, Kohntop R, Hasel C, et al. Evidence of Notch pathway activation in the ectatic ducts of chronic pancreatitis. J Pathol. 2008;214:312–319
  34. Kayed H, Kleeff J, Keleg S, et al. Distribution of Indian Hedgehog and its receptors patched and smoothened in human chronic pancreatitis. J Endocrinol. 2003;178:467–478
  35. Lowenfels AB, Maisonneuve P. Epidemiology and risk factors for pancreatic cancer. Best Pract Res Clin Gastroenterol. 2006;20:197–209
  36. Malka D, Hammel P, Maire F, et al. Risk of pancreatic adenocarcinoma in chronic pancreatitis. Gut. 2002;51:849–852
  37. Thayer SP, di Magliano MP, Heiser PW, et al. Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature. 2003;425:851–856
  38. Berman DM, Karhadkar SS, Maitra A, et al. Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours. Nature. 2003;425:846–851
  39. Guerra C, Mijimolle N, Dhawahir A, et al. Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context. Cancer Cell. 2003;4:111–120

 Supported by the “Ramón y Cajal” program from the Spanish Ministry of Science and Education (D.C.) and grants from the NIH (DK60533, CA112537 to M.H.).

PII: S0016-5085(08)01184-0

doi:10.1053/j.gastro.2008.06.063

Refers to article:

  • Editorial Accompanies this ArticleAdditional Online Content Available Hedgehog Signaling Is Required for Effective Regeneration of Exocrine Pancreas , 17 April 2008

    Volker Fendrich, Farzad Esni, Maria Veronica R. Garay, Georg Feldmann, Nils Habbe, Jan Nygaard Jensen, Yuval Dor, Doris Stoffers, Jan Jensen, Steven D. Leach, Anirban Maitra
    Gastroenterology August 2008 (Vol. 135, Issue 2, Pages 621-631.e8)

Gastroenterology
Volume 135, Issue 2 , Pages 347-351, August 2008