The underlying mechanism of acute pancreatitis has long been thought to involve autodigestion of the pancreas by its own digestive proteases. Under physiological conditions, the pancreas is protected by a variety of mechanisms that include storage and processing of digestive enzymes in membrane-confined vesicles, transport of proteases to the lumen as inactive precursor zymogens, the presence of protease inhibitors and the absence of the physiological activator enterokinase from the pancreas. Several studies have shown that these protective mechanisms are apparently overwhelmed in the early phase of pancreatitis and protease activation, specifically the premature and intrapancreatic activation of trypsinogen, is an inherent characteristic of human and several experimental models of pancreatitis15,20,23
One well documented mechanism that permits the protease activation cascade to begin within acinar cells is the activation of trypsinogen by cathepsin B (CTSB). Several studies have shown with purified or recombinant enzymes4,8
, with isolated preparations of pancreatic acini, or with animal models of pancreatitis5,15
that CTSB is a potent activator of trypsinogen and its deletion or inhibition greatly reduces intrapancreatic protease activation and the severity of pancreatitis. A prerequisite for the activation of trypsinogen by CTSB is supposed to be a redistribution of lysosomal enzymes into the zymogen-containing secretory compartment and a colocalization of CTSB with trypsinogen. Both conditions are met when pancreatitis begins in either the human or animal pancreas5
. Whether other lysosomal proteases can have similar functions in the pancreas or in pancreatitis was previously unknown.
In the present study we found that cathepsin L (CTSL), the second most common lysosomal cysteine proteinase besides CTSB, is abundantly present in human and mouse pancreatic acinar cells. We further found that CTSL and CTSB are both localized in the lysosomal as well as the pancreatic secretory compartment and that their colocalization with zymogens further increases during pancreatitis and may even spread to the cytosol. Deletion of the ctsl gene, which does not affect the pancreas under physiological conditions, has two effects in experimental pancreatitis: 1) it greatly increases intrapancreatic trypsin activity because CTSL is a trypsin(ogen) inactivator and thus an antagonist of CTSB and 2) it greatly reduces the severity of pancreatitis possibly by shifting the cellular effects of pancreatitis towards apoptosis.
CTSB and CTSL are widely expressed members of the papain family of cysteine proteinases9
. Recent experimental results suggest that they not only catalyze bulk proteolysis but also take part in proteolytic processing of protein substrates9
. For CTSB, we and others have shown that it acts as a trypsinogen-activating enzyme in vivo and in vitro4,5
and that this process involves the cleavage of a Lys-Ile bond releasing active trypsin and the pro-peptide TAP. Due to its optimum at a pH <6, CTSB exerts its catalytic activity in an acidic intracellular compartment, where also zymogen activation has been shown to take place20
. We found that CTSL, the second most abundant cysteine proteinase, shares the same intracellular compartment and confirmed24
that it possesses a much higher endoproteolytic activity than CTSB.
We further found that CTSL very effectively cleaves trypsinogen at position G26-G27 resulting in its inactivation. Human cationic trypsinogen was found to be cleaved at the exact same position. This cleavage site is located three amino acids towards the C terminus from the physiological (i.e. for enterokinase and CTSB) cleavage-site Lys-Ile and removes the N terminus of mature trypsin, thus impairing the catalytic center of trypsin25
. Incubation of mature trypsin with CTSL, on the other hand, resulted in a negligible loss of the (Ile-Val-)-N terminus. This indicates a conformational change of trypsinogen upon physiological cleavage of TAP by either CTSB or autoactivation which prevents further processing of the N terminus by CTSL.
We also identified an additional cleavage site for CTSL at position E82-G83. This cleavage site is located in the calcium-binding loop and affects trypsinogen as well as trypsin. Under conditions at which trypsin(ogen) binds Ca++, e.g. pH 5.5, the cleavage by CTSL is strongly suppressed. On the other hand, EDTA-removal of Ca++ or decreasing pH to 4.0 strongly enhanced trypsin(ogen) degradation. Consistent with these findings the initial inactivation mechanisms for trypsinogen or trypsin by CTSL follow different pathways: i) trypsinogen is primarily cleaved in the N-terminal region in a Ca++-independent manner (G26-G27), ii) the primary cleavage of trypsin occurs predominantly at the Ca++-binding site (E82-G83). Which of these two prevails in vivo is impossible to predict but during experimental pancreatitis the absence of CTSL results in a manifold increase in intrapancreatic trypsin activity.
The proteolytic processing of trypsinogen by enterokinase or CTSB produces active trypsin and the cleaved pro-peptide in equal stoechiometric amounts. The amount of trypsinogen activation peptide (TAP) therefore reflects the extent of trypsinogen activation much more accurately than activity measurements, e.g. when trypsin is rapidly inactivated by autodegradation23
, endogenous inhibitors, or proteolytic degradation. Due to its relative stability and the availability of specific antibodies, TAP is increasingly recognized as a standalone parameter for pancreatitis severity and has found its way into clinical practise23
. Our results now indicate a second pathway in which the generation of trypsin activity and TAP do not develop in parallel. We found that the primary cleavage of trypsinogen by CTSL creates a TAP-IVG peptide that escapes detection by the TAP antibody. However, its subsequent conversion by CTSB produces immunoreactive TAP to a much greater extent than via direct CTSB activation of trypsinogen. Obviously, this pathway combines the highly effective endoproteolytic activity of CTSL with the exoproteolytic activity of CTSB. In the pathological situation of acute pancreatitis, these large amounts of TAP may no longer reflect trypsin activity.
Our experiments, using isolated pancreatic acini with either specific enzyme inhibitors or from CTSL and CTSB knockout animals, confirmed this fundamental difference between the two lysosomal hydrolases. CTSB was confirmed as an activator of trypsinogen and of the intracellular protease cascade, whereas CTSL was identified as its antagonist and a potent inactivator of trypsinogen and trypsin. Taken together, these data suggest that alterations in the structure or function of CTSL could represent an important mechanism in the pathogenesis of human pancreatitis.
The effect of CTSL on disease severity seems to be completely uncoupled from its effect on trypsinogen activation. It not only affects pancreatic injury directly but also translates into less systemic inflammation, even in a model such as taurocholate-induced pancreatitis in which trypsinogen activation might not play such a crucial role in determining disease severity. It is also paralleled by increased caspase-3 activation, PARP cleavage and apoptosis in the pancreas of Ctsl−/−
animals. That such a shift to apoptosis-dominant cell injury improves the outcome of pancreatitis has previously been reported26
. Cathepsins, on the other hand, have long been considered to be guardians of the cellular homeostasis and, when released into the cytoplasm, to influence apoptotic pathways through a number of different mechanisms27
. A release of cathepsins into the cytoplasm has recently been shown to also occur in pancreatitis28
. Crossbreeding of Ctsl−/−
mice results in a lethal phenotype around postnatal day 12 and is caused by significant neuronal cell apoptosis, whereas neither the CTSL nor the CTSB knock-out alone has such an effect. This suggests that some in vivo
function of CTSB can be compensated for by CTSL and vice versa29
. Other experimental observations would also be in line with a predominantly antiapoptotic role of CTSL30,31
. In the setting of pancreatitis, however, this antiapoptotic function of CTSL appears to contribute to disease severity and the absence or inhibition of CTSL would have a beneficial therapeutic effect.
In conclusion, the present study establishes CTSL as a potent trypsin(ogen)-inactivating factor in vivo and in vitro and thus as an antagonist of CTSB in the digestive protease cascade that triggers pancreatitis. The antiapoptotic function of CTSL, on the other hand, affects disease severity in a manner that is unrelated to the initiating protease cascade. As far as the understanding of acute pancreatitis is concerned, these data indicate further that: i) greater intrapancreatic trypsin activity does not necessarily correlate with greater acinar cell injury, and ii) higher TAP levels do not always reflect higher levels of active trypsin or disease severity. The sequence in which lysosomal proteases find their substrates, the subcellular compartment in which they either constitutively or pathophysiologically colocalize with digestive enzymes, as well as the biophysical properties of that compartment ultimately determine whether their action is disease provoking or protective.