SONIC HEDGEHOG PROCESSING, SECRETION AND FUNCTION IN THE STOMACH

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1 Page 1 of 19 Articles in PresS. Am J Physiol Gastrointest Liver Physiol (February 28, 2008). doi: /ajpgi SONIC HEDGEHOG PROCESSING, SECRETION AND FUNCTION IN THE STOMACH Yana Zavros Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, OH. Correspondence to: Yana Zavros, Ph.D. University of Cincinnati College of Medicine Department of Molecular and Cellular Physiology 231 Albert B. Sabin Way 4259A, MSB Tel: (513) Fax: (513) yana.zavros@uc.edu Abbreviation: Sonic Hedgehog (Shh), Hedgehog (Hh), Indian Hedgehog (Ihh), Desert Hedgehog (Dhh), Hedgehog receptor patched (Ptch) Key Words: parietal cells, pepsinogen, cancer, inflammation, H. pylori 1 Copyright 2008 by the American Physiological Society.

2 Page 2 of 19 ABSTRACT Sonic Hedgehog (Shh) is recognized as one of the main morphogens that regulates cell differentiation during early development of the stomach. In the adult stomach, Shh is expressed and secreted from the acid-producing parietal cells, where it is believed to play an essential role in gastric tissue homeostasis and normal differentiation of the epithelium. The current themes article has focused on reviewing the literature and controversies surrounding the processing and secretion, and the role of Shh in the adult stomach. 2

3 Page 3 of 19 INTRODUCTION The role of Hedgehog in the genetic control of developmental patterning was first elucidated by Nüsslein-Volhard and Wieschaus where the effect of mutations on the segmented Drosophila (fruit fly) embryo, using a saturation mutagenesis screen, was studied (11). It was shown that in the wild type Drosophila embryos bristles covered the anterior half of each segment, while the posterior remained smooth. In contrast to the wild type Drosophila larvae, Nüsslein-Volhard and Wieschaus found a group of mutants whereby the posterior half of each segment failed to develop, resulting in the embryos covered entirely with bristles (11). The bristled phenotype resembled that of a hedgehog from which the name originated. Since the identification of the Hedgehog mutant, three hedgehog homologs have been described that include Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh). Although it is known that Shh is highly expressed in the stomach, it is only recently that the regulation of its expression and secretion have been investigated. Shh is expressed in adult stomach where it is believed to play a role as a regulator of gastric epithelial differentiation. The strongest evidence suggesting a pivotal role for Shh in the development of the stomach comes from Shh null mice that show an intestinal rather than gastric-type mucosa (9, 15). In addition, pharmacological inhibition of the Shh signaling pathway in the adult stomach causes loss of parietal cells (gastric atrophy) and subsequent disruption of glandular differentiation (21). Emerging studies reveal that Shh is involved in parietal cell function and maintenance of gastric epithelial differentiation. However, the 3

4 Page 4 of 19 precise role of Shh as a regulator of gastric epithelial cell differentiation or function is largely unknown. SONIC HEDGEHOG PROCESSING IN THE MAMMALIAN STOMACH Shh is synthesized as a full-length precursor protein that migrates with a relative mass of approximately 45 kda. It has been shown previously that the precursor protein undergoes an autocatalytic cleavage reaction to generate a biologically active amino-terminal polypeptide corresponding to an approximate 19 kda fragment. The amino-terminal domain (ShhN) is the active signaling peptide. During the cleavage process, a cholesterol moiety is added to the carboxyterminus of ShhN (ShhNp, 26kDa) which then remains membrane tethered. A multimeric form of ShhN is secreted, soluble and freely diffusible (reviewed in (6)) (Figure 1). Either the cell retained or secreted forms of Shh are able to bind to the hedgehog receptor patched (Ptch) to activate Hh signaling (reviewed in (6)). Studies demonstrating autocatalysis of Shh protein have been performed using either Drosophila or zebrafish models (13). In both models, hedgehog proteins undergo proteolytic processing in the absence of microsomal membranes (13). In contrast, processing of mammalian Shh protein translated in vitro fails unless microsomal membranes are added (1). Interestingly, in vitro experiments using a gastric cancer cell line, having similar characteristics to parietal cells (23132/87 cells), showed that Shh transcription is induced by low ph conditions in culture media (3). This evidence suggests that, at least in mammalian systems, processing of Shh protein requires specific cellular factors. 4

5 Page 5 of 19 Recently, we have found that in the stomach Shh processing occurs via a hormonally regulated and acid-dependent mechanism (24). In addition to regulating acid secretion, gastrin acts as a growth factor for the gastric mucosa. An infusion of gastrin for fourteen days resulted in a significant stimulation of both Shh expression and processing. Moreover, in vitro studies revealed that processing of the 45 kda Shh precursor protein occurred under acidic conditions and was cleaved by the acid-activated protease pepsin A (24) (Figure 1). Consistent with our findings was a recent publication by El-Zaatari et al (4) demonstrating that during hypergastrinemia and elevated acid secretion, there is a concomitant increase in Shh expression. Thus, evidence from these studies show for the first time that production of the biologically active Shh protein depends upon stimulation of gastric acidity. SONIC HEDGEHOG EXPRESSION AND SECRETION FROM THE GASTRIC MUCOSA The expression of Shh within the gastric mucosa is controversial. While it is shown by a number of laboratories that Shh is expressed and secreted from parietal cells (18, 21, 24), protein expression in the pit cells is unclear. One study has detected Shh expression in the pit region of Mongolian gerbils using in situ hybridization (19), but in the mouse stomach there is conflicting data. While there is report of an absence of Shh expression in pit cells of the mouse gastric mucosa (21), another study confirms the Shh expression in this region (4). The discrepancy has been attributed to differences between mouse strains, specificity 5

6 Page 6 of 19 of antibodies and sensitivity of methods used to detect Shh expression. The reason for the discrepancy remains unclear. Despite the discrepancy of Shh expression within the pit cells of the gastric mucosa, it is clear that Shh is expressed and secreted from the acid-producing parietal cells (18, 24). Emerging evidence from our lab suggests that both processing and secretion occur at the apical membrane of parietal cells (Zavros et al., Manuscript Under Review). In support of apical expression of Shh, green fluorescent protein (GFP) fused with the C-terminus of the human Shh protein transfected into polarized 293T cells revealed that expression of Shh protein was concentrated at the lateral and apical surfaces of the plasma membrane, areas that are enriched in cholesterol and the likely site of posttranslational modification (22). Interestingly, the apical secretory membrane of the parietal cell also contains a high lipid content that is critical for selective permeability of water and ions, but not urea or ammonia. One working hypothesis is that the Shh 45 kda precursor may be translocated to the apical membrane during tubulovesicular fusion and processed at the canalicular membrane where activated H +,K + - ATPase has been recruited, acid is secreted and pepsinogen A is converted to pepsin A (Figure 2). SONIC HEDGEHOG FUNCTION IN THE ADULT STOMACH Shh is believed to regulate epithelial cell differentiation in the adult stomach, but its role as a morphogen is based on evidence that correlates the loss of Shh with neoplastic transformation of the gastric mucosa (17, 21). Although Shh null 6

7 Page 7 of 19 mice have given us insight into the importance of this protein in the development of the gastric epithelium, deletion of the Shh gene is embryonic lethal (9, 15) making it impossible to study the function of Shh in the adult stomach under physiological conditions. Moreover, data from the gastrin-deficient mice demonstrate that gastrin may be a trophic factor for the gastric epithelium through its activation of Shh secretion (24), but the direct effect of Shh cannot be tested in this model. Certainly, secretion of Shh across the apical membrane of parietal cells would suggest that Shh binds to its receptor Ptch to behave as a lumicrine growth factor for the gastric epithelium. During development of the gastrointestinal tract, there are a number of examples of luminal growth factors that are involved in morphological and functional differentiation. For example, luminal EGF and TGF regulate the maintenance of epithelial renewal (7), and in the intestine and likely stomach, trefoil peptides promote restitution following secretion onto the apical surface (12). Whether Shh acts as a lumicrine growth factor for gastric epithelial cell differentiation remains to be determined In support of a lumicrine function for Shh, t has recently been discovered that the Ptch receptor delocalizes from the cell membrane once Shh is bound as a Ptch-Shh complex on primary cilia. Primary cilia are projections found on almost all vertebrate cell surfaces that extend into the extracellular environment and function as sensors for various signals, including changes in Shh levels (16). From these findings we may hypothesize that ciliated cells may be capable of sensing changes in luminal or apically secreted Shh. 7

8 Page 8 of 19 Besides its proposed role as a growth factor for the gastric epithelium, Shh may also be a fundamental regulator of the physiological regulation of acid secretion. We know that Shh regulates H+,K+-ATPase expression in canine parietal cells (18). Moreover, treatment of mice with the hedgehog signaling inhibitor cyclopamine results in elevated circulating gastrin levels, suggesting that Shh may be an important factor in the regulation of gastrin secretion (4). Gastrin, histamine and acetylcholine are the major secretagogues of gastric acid secretion. Increased acidity then stimulates chemoreceptors on the D-cells to secrete somatostatin and block further release of gastrin and gastric acid. Thus, gastric acid secretion is regulated by a negative feedback mechanism. Certainly, the hypergastrinemia observed with cyclopamine treatment (4) may suggest that blockade of Shh activity results in the removal of the somatostatin-inhibitory effect on gastrin. The possible role of Shh as a mediator of such a crucial regulatory mechanism of acid secretion in the stomach is an area that requires further investigation. PEPSINOGEN A AND SONIC HEDGEHOG - MARKERS OF PARIETAL CELL ATROPHY The stomach is not only the site of acid secretion, but also the major source of factors, including TGF, Wnt, FGFs and hedgehog proteins, that are responsible for regulating the differentiation of the gastric epithelium. Thus, it is not surprising that gastric atrophy and disruption of normal cell proliferation and differentiation triggers a cascade that develops into neoplasia (2). Prior to the develop- 8

9 Page 9 of 19 ment of dysplasia, the gastric mucosa is typically atrophic in which the acidproducing parietal cells are drastically reduced or absent. Analysis of human stomachs has shown that the presence of gastric atrophy is a reliable indicator of pre-neoplastic changes in the stomach (2). Loss of mature parietal cells from the body of the stomach by either genetic or pharmacologic methods results in severe abnormalities in the differentiation and development of gastric cell lineages characterized by the appearance of intestinal or gastric metaplastic cells (8). Interestingly, changes in a number of proteins or physiological functions have been reported as indicative or as markers of atrophy. These include changes in serological pepsinogen A to C ratio, loss of acid secretion, hypergastrinemia (elevated plasma gastric concentrations), suppressed somatostatin expression, loss of E-cadherin (protein involved in cell-adhesion) (10) and more recently loss of Shh (17). It is widely accepted that inflammation that is caused by Helicobacter pylori (H. pylori) infection is a trigger for the development of gastric cancer (2). In fact, exogenous infusion of only IFN into mice is sufficient to induce significant mucous gland metaplasia and hypergastrinemia (25). However, the question of the mechanism by which inflammatory cytokines induce mucosal damage remains unanswered. Given that loss of Shh may be predicted to be an initial marker for the development of gastric cancer, there is surprisingly little information regarding the regulation of Shh by pro-inflammatory cytokines. In a Mongolian gerbil model of H. pylori infection, it has been shown that bacterial colonization leads to downregulation of Shh expression (19), and in humans, loss of Shh may be an early 9

10 Page 10 of 19 change that occurs in the mucosa prior to neoplastic transformation (17). Therefore, loss of Shh expression has been correlated with intestinal metaplasia and may play a role in carcinogenesis. However, the mechanism that results in loss of Shh in the infected gastric mucosa is unexplored. If gastric acidity is reduced, due to inhibition of acid secretion by inflammatory cytokines, then pepsinogen A is not activated and we would hypothesize that there would be loss of Shh function due to either loss of processing or secretion. This notion is consistent with the observation that gastric atrophy correlates with reduced pepsinogen A to C ratios in stomachs of patients with increased risk of cancer development (17). Numerous studies have evaluated the ratio of Pepsinogen A to Pepsinogen C in the assessment of a patient s increased risk for developing gastric cancer (10). Low levels of Pepsinogen A are predictive of chronic atrophic gastritis, which is the most important predictor of gastric transformation (10). Certainly, our data demonstrate that in tumor tissue from human stomach, Pepsinogen A expression is significantly lower compared to normal gastric mucosa. Moreover in tumor tissue, Shh processing is lost similar to that of normal extracts immunodepleted of Pepsinogen A (24). Collectively, these findings underscore the importance of Shh in the maintenance of normal gastric mucosal differentiation in the corpus. Given that an acid environment is translatable to other cells, the processing mechanism for Shh also gives us new insight into the Shh expression in other systems. For example, a number of studies show that another member of the aspartic protease family cathepsin D is involved in osteoclastogenesis (23). Cer- 10

11 Page 11 of 19 tainly, this setting suggests a favorable environment for the activation of the aspartic protease family that includes pepsin A, C and cathepsins, but their role as processing enzymes for Shh during development are unknown. Generally, members of the aspartic protease family are also increased in the tumor tissues that are highly acidic and intriguingly rich in Shh protein (14). In both the setting of either tissue development or cancer, the ubiquitous expression of this family of proteases in different tissues suggests that regulation of Shh processing may not be limited to the stomach. SONIC HEDGEHOG AND GASTRIC CARCINOGENESIS The role of Hh signaling in the development of gastric cancer has become evident from studies of both various gastrointestinal cancer cell lines and analysis of patient gastric samples (17, 20). A study by Berman et al (Berman et al., 2003, reviewed in (20)) clearly demonstrated elevated Hh signaling activity, supported by increased Ptch receptor expression, occurred in gastric carcinoma. The over-activity of the Hh pathway during carcinoma was explained by overexpression of the Hh ligand that was blocked by both cyclopamine (hedgehog signaling antagonist) and a Hh blocking antibody (Berman et al., 2003, reviewed in (20)). Conversely, when patients with evidence of pre-cancerous lesions such as atrophy and intestinal metaplasia are studied, there is a strong loss of Shh protein expression (17). Interestingly, a recent study showed that during pseudopyloric metaplasia, a lesion induced by Helicobacter infection, resulted in the reactivation of Shh (5). It may be that such conflicting data indicates that Hh sig- 11

12 Page 12 of 19 naling is active in the later stages of gastric carcinogenesis in response to inflammatory cytokines. There may also be differences in the expression of Shh and tumor subtype. Testing Hh pathway activation in a cohort of patients with gastric cancer would address the role of Hh ligand production and expression in tumors. This also underscores the need for genetically modified mice to study the role of Hh signaling in tumorigenesis. SUMMARY Recent studies have shown that processing of Shh within the adult stomach is dependent upon acid secretion from parietal cells. In the stomach Shh plays a crucial role in parietal cell function and possibly the regulation of epithelial cell differentiation. Thus loss of Shh during atrophic gastritis, a known precursor lesion to gastric cancer, is clinically significant. Unfortunately, our understanding of Shh in the adult stomach has been disadvantaged by the embryonic lethality of the hedgehog signaling mutant models. The use of cell-specific inducible hedgehog signaling mutants will be critical to revealing the mechanisms by which Shh regulates epithelial cell homeostasis in the stomach. FIGURE LEGENDS Figure 1: Schematic diagram of Shh processing: Autocatalytic versus protease-dependent mechanisms. The cleavage of the 45kDa full length/nascent peptide generates the signal peptide (39kDa). Autocatalytic cleavage at the Gly- 12

13 Page 13 of 19 Cys residues yields a processed and secreted 19kDa peptide that is lipid modified (ShhNp). In the parietal cell Shh undergoes processing by an acid- and protease-dependent mechanism. Processing via the protease dependent mechanism generates a 19kDa freely secreted protein (ShhN). The predicted site of pepsin A cleavage is at the Cys-Phe residues. Figure 2: Expression and processing of Shh at the apical membrane of gastric parietal cells. Based on the common location of Shh and H +,K + - ATPase, processing may occur at the apical membrane of the parietal cell after stimulation. Shh protein present within tubulovesicles of resting parietal cells traffic to the apical membrane upon stimulation of the parietal cell and acid secretion. The protein is then accessible to the active pepsin A enzyme where Shh is processed and secreted across the apical membrane. ACKNOWLEDGEMENTS I would like to thank Dr. DJ Robbins (Dartmouth Medical School) for his helpful discussions during the preparation of this article. I would also like to acknowledge the expert assistance of Glen Doerman with Figure 2B. This work was supported by start-up funds from the Department of Molecular and Cellular Physiology, University of Cincinnati (Y.Z.). 13

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19 Page 19 of 19 Figure 2 Shh 19kDa secreted across the apical membrane ATP ATP ATP ATP H+ AT P ATP H+ K+ : Pepsin A : Shh 45kDa : Shh 19kDa ATP : H+,K+-ATPase : Tubulovesicles ATP H+ H+ ATP K+ H+ H+