Hereditary Pancreatitis: 25 Years of an Evolving Paradigm

Abstract

The identification of the genetic basis of hereditary pancreatitis in 1996 confirmed the critical role of trypsinogen in this disease and opened a new avenue of research on pancreatitis-associated genetic risk factors and their mechanism of action. Through the following 25 years, the ensuing discoveries fundamentally changed our understanding of pancreatitis pathogenesis, clarified the role of trypsinogen autoactivation in disease onset and progression, and set the stage for future therapeutic interventions. This Frank Brooks Memorial Lecture was delivered on November 4, 2021, at the 52^nd Annual Meeting of the American Pancreatic Association, held in Miami Beach, Florida.

Almost a quarter of a century ago, the renowned pancreatologist Michael Steer gave the Frank Brooks Memorial Lecture at the annual meeting of the American Pancreatic Association.¹ In his treatise, entitled “The early intraacinar events which occur during acute pancreatitis”, he described the mechanism of intrapancreatic trypsinogen activation by cathepsin B (CTSB) as the critical initiating event in the development of pancreatitis. The generally accepted hypothesis at the time posited that pancreatitis was elicited by acinar cell injury that would lead to pathological colocalization of lysosomes (which contain the cysteine protease CTSB) and zymogen granules (filled with the trypsin precursor trypsinogen). Cathepsin B catalyzes activation of trypsinogen to trypsin, which, in turn, would cause acinar cell necrosis and consequent inflammation. The colocalization theory of pancreatitis pathogenesis and the central role of CTSB-mediated trypinogen activation has remained a prevailing doctrine ever since.^2–5 Somewhat ironically, only a year before the Steer lecture, historic discoveries were made that would revitalize research and eventually change our understanding of pancreatitis pathogenesis. Thus, in 1996 three laboratories mapped the hereditary pancreatitis gene to chromosome 7, and David Whitcomb and co-workers identified the serine protease 1 (PRSS1) gene mutation p.R122H as a causative genetic defect in this inherited disorder.^6–9

THE BIOCHEMISTRY OF HEREDITARY PANCREATITIS

The PRSS1 gene codes for human cationic trypsinogen. Because mutations in trypsinogen cause pancreatitis and activation of trypsinogen by CTSB triggers pancreatitis, it was presumed that the mutations might stimulate CTSB-mediated trypsinogen activation.¹⁰ Surprisingly, biochemical studies failed to confirm this notion and showed that most PRSS1 mutations had either no impact on the activation reaction or even inhibited it.^11–13 A striking example of the latter was mutation p.D22G, which abolished trypsinogen activation by CTSB.^12,13 Remarkably, however, mutation p.D22G and other PRSS1 mutations located in the trypsinogen activation peptide (eg. p.K23R) robustly stimulated autoactivation of trypsinogen,^14–18 suggesting that the pathomechanism of hereditary pancreatitis involves increased autoactivation rather than increased CTSB-mediated trypsinogen activation. Autoactivation is the common biochemical term for the reaction in which trypsin activates trypsinogen in a self-amplifying manner. The validity of the autoactivation hypothesis, however, was challenged by the fact that the clinically more frequent PRSS1 mutations, such as p.N29I and p.R122H, had only a minor impact on atoactivation.^19,20 This conundrum was resolved by a series of exciting studies that demonstrated that autoactivation of human cationic trypsinogen is regulated through proteolytic cleavages by chymotrypsin C (CTRC).^20–25 We discovered two seemingly opposing regulatory pathways; CTRC cleavage after Phe18 in the activation peptide stimulated autoactivation while cleavage after Leu81 in the calcium binding loop promoted degradation (Fig. 1). The predominant effect of CTRC is trypsinogen degradation. Importantly, we found that hereditary pancreatitis associated mutations interfered with the CTRC-dependent regulation by either blocking or reducing CTRC-mediated degradation (eg. p.R122H),^20,22,25 or by increasing the stimulatory effect of CTRC on autoactivation (eg. p.A16V).^20,21,25 Taken together, biochemical studies convincingly demonstrated that pancreatitis-associated PRSS1 mutations increase autoactivation of human cationic trypsinogen either in a CTRC-dependent manner or directly, independently of CTRC (Fig. 1).

FIGURE 1.

Mechanism of PRSS1-related hereditary pancreatitis. Mutations p.N29I, p.N29T, p.V39A, p.R122C, and p.R122H block or reduce chymotrypsin C (CTRC)-mediated trypsinogen degradation. Mutations p.A16V, p.P17T, and p.N29I promote CTRC-dependent stimulation of autoactivation. Activation peptide mutations p.D19A, p.D21A, p.D22G, p.K23R, and p.K23_I24insIDK (p.insIDK) directly accelerate trypsinogen autoactivation, independently of CTRC. All PRSS1 mutations indicated result in more robust autoactivation of trypsinogen to active trypsin. Figure modified from reference.²⁴

MOUSE MODELS OF HEREDITARY PANCREATITIS

To model the effect of PRSS1 mutations in mice, we generated two mouse lines, T7D23A and T7K24R, which harbor mutations in the activation peptide of mouse cationic trypsinogen (isoform T7).²⁶ The T7D23A mice carry the p.D23A mutation (Fig. 2),²⁷ which is analogous to the human p.D22G mutation in PRSS1. Note that the activation peptide of cationic trypsinogen has an extra Asp residue in the mouse, which shifts amino acid numbering by one relative to the human isoform. In our model design, we changed the Gly residue found in the p.D22G human mutation to Ala in the T7D23A mice to accentuate the phenotype. The p.D23A mutation increases autoactivation of mouse cationic trypsinogen by 50-fold while it has no effect on CTSB-mediated trypsinogen activation. Heterozygous T7D23A mice develop spontaneous acute pancreatitis as early as 3 weeks of age with the classic signs of high plasma amylase activity, pancreas edema, inflammatory cell infiltration, and scattered acinar cell necrosis. Progression to chronic pancreatitis is rapid and by 1–2 months of age mice exhibit pancreas atrophy with acinar cell ablation, regenerative pseudotubular complexes, dilated ducts, diffuse fibrosis, and persistent inflammatory cell infiltration (Fig. 3). End-stage disease is apparent between 6 and 12 months of age and it is characterized by severe pancreas atrophy, dilated ducts, adipose infiltration, and an abundance of enlarged islets of Langerhans. The disease phenotype of T7D23A mice provided compelling evidence that markedly increased trypsinogen autoactivation can drive onset of acute pancreatitis with subsequent progression to chronic pancreatitis.

FIGURE 2.

Amino-acid sequence of the trypsinogen activation peptide in mouse cationic trypsinogen (T7) and human cationic trypsinogen (PRSS1). The p.D23A and p.K24R mutations used in the T7D23A and T7K24R mouse models and the corresponding p.D22G and p.K23R human PRSS1 mutations are indicated. The same activation site is cleaved by trypsin and cathepsin B. Note that amino-acid numbering in mouse T7 trypsinogen is shifted by one relative to human PRSS1. Figure modified from reference.²⁵

FIGURE 3.

Spontaneous acute pancreatitis and progression to chronic pancreatitis in T7D23A mice.²⁶ Representative hematoxylin-eosin stained pancreas sections are shown. The scale bars correspond to 100 μm. Figure modified from reference.²⁵

The T7K24R mice carry the p.K24R mutation (Fig. 2),²⁸ which corresponds to the human p.K23R mutation in PRSS1. The mutation accelerates autoactivation of mouse cationic trypsinogen by 5-fold but causes no increase in CTSB-mediated trypsinogen activation. Thus, the propensity for the mutant trypsinogen to autoactivate is significantly lower in T7K24R mice than in T7D23A mice. As a result, even homozygous T7K24R mice are phenotypically normal and develop no spontaneous pancreatic disease, unlike T7D23A mice. However, when challenged with a supramaximal stimulatory dose of cerulein, T7K24R mice exhibited increased intrapancreatic trypsin activation and developed more severe acute pancreatitis, as judged by higher plasma amylase activity, increased pancreas edema, and stronger inflammatory cell infiltration, relative to C57BL/6N mice.²⁸ Remarkably, when the natural course of cerulein-induced pancreatitis was compared in T7K24R and C57BL/6N mice, we found that T7K24R mice developed progressive chronic pancreatitis with acinar cell atrophy, persistent macrophage infiltration, and diffuse fibrosis (Fig. 4).²⁹ Histological recovery was markedly delayed and permanent, chronic changes were still detectable 1–3 months after the acute pancreatitis episode. In contrast, C57BL/6N mice rapidly recovered after an acute episode of pancreatitis and achieved complete histological restitution within 3 days. Taken together, the pathological phenotype of T7K24R mice indicate that moderately increased trypsinogen autoactivation sensitizes the pancreas to injury and results in more severe acute pancreatitis followed by chronic disease progression.

FIGURE 4.

Chronic progression and delayed recovery after an episode of cerulein-induced acute pancreatitis in T7K24R mice.²⁸ Histological recovery of C57BL/6N mice after an episode of acute pancreatitis is shown for comparison. Representative hematoxylin-eosin stained pancreas sections are presented. The scale bars correspond to 100 μm.

The mouse models of hereditary pancreatitis confirm that trypsinogen mutations are directly pathogenic, and the extent of increased autoactivation determines pancreatitis responses and pathology. More robust autoactivation (T7D23A model) causes spontaneous pancreatitis whereas a smaller increase in autoactivation (T7K24R model) results in severe and progressive experimental pancreatitis. Importantly, both models can serve as invaluable tools for preclinical testing of drugs targeting intrapancreatic trypsin.

THE ROLE OF CATHEPSIN B IN HEREDITARY PANCREATITIS

Human genetic studies failed to find any credible association between CTSB variants and hereditary or idiopathic chronic pancreatitis. A common CTSB variant was suggested to increase pancreatitis risk slightly in Indian cohorts, however, this finding could not be replicated in a European cohort.^30,31 Furthermore, unbiased genome wide association studies did not reveal a link between chronic pancreatitis and CTSB variants either.^32,33 Biochemical and mouse modeling studies demonstrated that increased autoactivation of mutant trypsinogens, rather than CTSB-mediated trypsinogen activation, underlies the pathogenesis of hereditary pancreatitis. Furthermore, a review of the published literature revealed several mouse studies showing that cerulein-induced intrapancreatic trypsin activity is not a determinant of pancreatitis severity. (I) Thus, global deletion of CTSB diminished cerulein-induced intrapancreatic trypsin activity but the mice still developed acute pancreatitis albeit with somewhat reduced severity relative to controls.³⁴ These observations were heralded as proof of the CTSB centric concept of pancreatitis pathogenesis and emphasis was placed on the modest reduction in disease severity rather than the fact that pancreatitis could develop in the absence of CTSB. (II) Among the other lysosomal cysteine proteases, cathepsin L (CTSL) also cleaves trypsinogen, however, this proteolytic event inactivates trypsinogen. Accordingly, genetic deletion of CTSL in mice resulted in a marked elevation of cerulein-induced intrapancreatic trypsin activity, but this increase did not translate to more severe disease.³⁵ In fact, experimental acute pancreatitis was ameliorated in CTSL-deficient mice. (III) Similarly, cerulein elicited high intrapancreatic trypsin activity with no change in disease severity in mice that missort CTSB to the secretory compartment due to deficiency in the cation-independent mannose 6-phosphate receptor.³⁶ (IV) Finally, genetic deletion of mouse cationic trypsinogen (T7 knockout) nearly abolished cerulein-induced intrapancreatic trypsin activity but the mice still developed acute and chronic pancreatitis with relatively small changes in severity.^37,38 Taken together, the studies cited indicate that cerulein-induced intrapancreatic trypsin activity in mice is due to the CTSB-mediated activation of trypsinogen. However, this epiphenomenon plays little, if any, role in the subsequent development of pancreatitis.

To address this problem in a more definitive manner, we designed a mouse cationic trypsinogen mutant which was defective in autoactivation yet showed increased activation by CTSB.³⁹ We achieved this by replacing the activation site Lys24 residue with Gly (p.K24G) and changing the preceding Asp22 residue to Ala (p.D22A) in the activation peptide. More recently, working with Andrea Geisz at Boston University, we generated a mouse model carrying these mutations. As expected from the biochemical studies, the mutant mice exhibited significantly increased cerulein-induced intrapancreatic trypsin activity compared to the C57BL/6N parent strain. Preliminary studies suggested that cerulein-induced pancreatitis severity and disease course is unaltered in these mice, which were engineered specifically for increased CTSB-mediated trypsinogen activation. Once finalized, these experiments will strengthen our notion that CTSB should no longer be considered a potential pharmacological target to treat or prevent pancreatitis. Instead, we need to refocus our preclinical efforts to mitigate autoactivation by targeting trypsinogen or trypsin.