Human pancreatitis and the role of cathepsin B

Short abstract

Any assumption about the role of the newly detected cathepsin B polymorphisms in pancreatitis must, for now, remain speculative

Acute pancreatitis has long been considered an autodigestive disorder in which the pancreas is destroyed by its own digestive proteases.¹ Under physiological conditions pancreatic proteases are synthesised as inactive precursor zymogens and stored by acinar cells in zymogen granules. Autodigestion of the gland would therefore require premature activation of these zymogens. How and where such a premature and intrapancreatic activation of digestive proenzymes is initiated in the course of pancreatitis has been the subject of several investigations.²,³ Recent studies strongly suggest that the early pathophysiological events that eventually lead to necrosis of pancreatic tissue originate in acinar cells³,⁴,⁵ and involve the intracellular presence of active trypsin,⁵,⁶ a serine proteinase capable of activating other pancreatic zymogens. Within pancreatic acinar cells cytoplasmic vesicles have been identified as the subcellular compartment in which premature trypsinogen activation begins within minutes after induction of experimental pancreatitis.³,⁷,⁸

The molecular mechanisms responsible for the intracellular activation of trypsinogen, however, have remained elusive. One hypothesis predicts that the lysosomal cysteine proteinase cathepsin B (CTSB) plays an essential role in this process.⁹ The largely circumstantial evidence for this “cathepsin B hypothesis” is based on the following observations: (a) CTSB has been shown to activate trypsinogen in vitro¹⁰; (b) during the initial phase of acute pancreatitis in several animal models, redistribution of CTSB into a zymogen granule containing subcellular compartment was detected by density gradient centrifugation¹¹; and (c) in the same pancreatitis models lysosomal enzymes were detected by immunogold electron microscopy in secretory organelles that also contained digestive enzymes (for example, trypsinogen).¹² Experimental approaches to show an essential role for CTSB in premature zymogen activation by inhibition of this lysosomal enzyme with synthetic inhibitors rendered contradictory results either increasing¹³ or decreasing premature zymogen activation,¹⁴ or failing to improve the course of experimental pancreatitis.¹⁵ To test the cathepsin B hypothesis more directly and to overcome the shortcomings of lysosomal enzyme inhibitors, which have only limited specificity for CTSB, a CTSB deficient mouse strain that was generated by targeted disruption of the ctsb gene was studied in an experimental animal model of pancreatitis.¹⁶ The results of these studies were unequivocal: 90% of intrapancreatic trypsinogen activation during pancreatitis depends on the presence of cathepsin B.¹⁶ While the reduction in local and systemic complications of pancreatitis that were conveyed by deletion of ctsb were not nearly as impressive, the experiments answered the question about the pathophysiological role of CTSB in premature digestive enzyme activation during experimental pancreatitis with a resounding yes and settled all arguments about this issue.

The relevance for human disease, however, is another matter. First attempts to establish the relevance of the cathepsin B‐pancreatitis hypothesis in humans focused on the capacity of the lysosomal enzyme to activate human trypsinogen, and specifically varieties of human trypsinogen, into which disease relevant mutations had been introduced that were identified in the context of hereditary pancreatitis studies. Hereditary pancreatitis is a type of pancreatitis that follows an autosomal dominant inheritance pattern, is associated with an early diseases onset of chronic pancreatitis (usually in children and young adults), and is associated with various germline mutations in the cationic trypsinogen (prss1) gene.⁵ When recombinant trypsinogen with hereditary pancreatitis mutations was subjected to activation by CTSB in vitro it was, indeed, found that some trypsins behaved differently from their wild‐type counterpart,¹⁷,¹⁸ an observation that clearly supported the cathepsin B hypothesis of pancreatitis. On the other hand, the most common PRSS1 mutations, such as R122H and N29I, did not convincingly vary from wild‐type trypsin in their activation kinetics by CTSB.¹⁹ The same study also demonstrated that CTSB is abundantly secreted from the human exocrine pancreas, plentifully contained in pancreatic secretory zymogen granules (rather than in lysosomes), as well as active within the secretory pathway.¹⁹ Thus all cellular conditions for the cathepsin‐B‐pancreatitis hypothesis to be operative in humans were met. Moreover, the proposed requirement for a subcellular redistribution of CTSB into the secretory compartment¹¹ could finally be put to test because most CTSB in the pancreas was found to already reside in the secretory compartment under physiological conditions¹⁹,²⁰ rather than having to be redistributed there from lysosomes. Nevertheless, no direct evidence for active involvement of CTSB in the onset of human pancreatitis—at least not in hereditary pancreatitis caused by the most common mutations—could be produced from these studies.

At this stage, Mahurkar and colleagues²¹ entered the fray with a study published in the present issue of Gut(see page 1270). This group from Hyderabad had been instrumental in characterising the genetic basis of tropical pancreatitis,²²,²³ a disease variety that was previously thought to be linked to dietary components (for example, cassava) or selenium deficiency and is now known to be linked to mutations in the pancreatic secretory trypsin inhibitor gene (spink1 gene).⁴ Because spink1 mutations explain only about half of the cases with tropical pancreatitis, Mahurkar et al sequenced the entire coding region of the ctsb gene from 51 South Indian patients with tropical pancreatitis and speculated that ctsb germline changes may explain the rest of cases. When they compared their ctsb sequencing data with that of 25 healthy controls they found 23 different polymorphisms and increased the number of patients to 140 (that of controls to 155) to genotype all of them for the four most interesting of these polymorphism. They found a significant difference between patients and controls only for a C76G polymorphism that results in a leucine to valine mutation at amino acid 26 (allele frequency in patients 0.46 versus 0.30 in controls). To rule out a chance finding, they went further south in India and recruited a second cohort of tropical pancreatitis patients (n = 166) and controls (n = 175) from Calicut and genotyped them for the same four polymorphism as the first group. Again, only the Leu26Val mutation was about twice as common among patients than controls.

So far the data would suggest that carrying a C76G polymorphism in the ctsb gene (that is, a leucine to valine mutation in the CTSB protein) would double the risk of developing tropical pancreatitis provided one is ethnically Drawidian and hail from southern India. The study then went further. The fact that the most common spink1 mutation associated with tropical pancreatitis (N34S) has no measurable effect on the trypsin inhibiting capacity of the SPINK1/PSTI protein²⁴ led to the speculation that, rather than causing tropical pancreatitis in India⁵ and idiopathic pancreatitis elsewhere,²⁵,²⁶ SPINK1 may act as a modifier gene for other genetic changes. That this was not the case for mutations in the cationic trypsinogen (prss1) gene had already been shown²⁷ but Mahurkar et al also tested it for ctsb. As found previously for trypsin, no differences in phenotype of pancreatitis or genotype with regard to N34S positive and N34S negative pancreatitis patients could be found for the L26V mutation. This demonstrates effectively that, whatever the effects of the CTSB mutations, they are unrelated to changes in SPINK1.

Other polymorphisms, those that do not lead to amino acid exchanges and were equally distributed between patients and controls, varied between N34S carriers and non‐carriers in further subgroup analyses but these may have been chance findings. Hard evidence was therefore only presented for a twofold pancreatitis risk in carriers or the L26V mutation in CTSB.

What could be the effect of this mutation at the cellular level? As no functional data are presently available, structural consideration must serve as a surrogate and the interpretation will necessarily remain speculative. The L26V mutation affects the propeptide region of CTSB which makes it very unlikely that it has an effect on the catalytic centre and thus on the enzymatic activity of CTSB. The most that could therefore be expected from a mutation at this site would be an effect on CTSB trafficking, but that may be sufficient to be disease relevant because it matters very much whether the mannose‐6‐phosphate dependent sorting of CTSB ends up in a lysosome or in a zymogen granule where dangerous substrates such as trypsinogens reside.

Little else can be learned from the actual polymorphism: a leucine to valine exchange is about as unexciting as a mutation can get, replacing one non‐polar amino acid with another that differs only in one CH₂ group. A more attractive explanation to emerge from functional studies would be that the mutation affects the capacity of CTSB to activate mesotrypsin, a trypsin variant that preferentially degrades SPINK1,²⁸ which in turn can inhibit cationic trypsin, which in turn has an established role in at least some variety of pancreatitis. As this complicated chain of events indicates, any assumption about the role of the newly detected ctsb polymorphism in the context of pancreatitis must remain wildly speculative. Even a role of CTSB in pancreatitis that is completely unrelated to activation of trypsin must be considered, just as the function of SPINK1 in the pancreas was found in knockout animal studies to involve embryonic pancreas development and not, to the surprise of many, the premature activation of trypsinogen during pancreatitis.²⁹

Functional studies that examine the biochemistry, cell biology, and interaction with other proteins for different CTSB variants will ultimately have to provide that answer. Whether other more common varieties of pancreatitis are equally associated with genetic ctsb changes will also have to be determined. Until then, Mahurkar et al have proven to us that: (1) the 30 year old cathepsin B hypothesis of pancreatitis has entered the phase in which evidence from human studies is finally being presented, and (2) that candidate gene sequencing based on pathophysiological information is still a valid and successful research strategy, particularly when well characterised patient cohorts are available for genotyping. The authors from Hyderabad should be congratulated on these achievements.