ADAM10 and 17 are metalloproteinases that modulate signalling in many physiological processes. For the liver regeneration, EGFR and c-Met pathways were identified as the most important ones. Several recent articles showed that only concurrent inactivation of both pathways causes inability of liver to recover from the partial hepatectomy and higher necrosis after CCl4 intoxication [11] [36] [7]. In this study, we demonstrate that both pathways are modulated by ADAM proteases in a pathological liver. Simultaneous hepatocyte-specific deficiency of both ADAM10 and ADAM17 led to lower activation of EGFR as well as to the lower shedding of a c-Met receptor. Thus, initial slower regenerative response after partial hepatectomy, caused by impaired shedding of EGFR activating ligands, was compensated by stronger HGF signalling through c-Met receptor. Furthermore we show, that both proteases are also involved in TNF RI release. ADAM17 directly cleaves the receptor, while ADAM10 deficiency causes elevation of TNF RI serum levels through an unidentified process.
Several EGFR ligands promote the proliferation of hepatocytes. It has been described that expression of HB-EGF, TGF-α and AREG is elevated after partial hepatectomy, even though they are not completely fungible [37]. ADAM17 has been described as the main protease in the release of TGF-α and AREG [27]. HB-EGF is shed mainly by ADAM17 and ADAM12, and betacellulin is a substrate of ADAM10 [27]. In addition, EGF [38] supplied to the liver from the duodenum, was identified as a substrate of ADAM10 [39]. We show that combined deletion of ADAM10 and ADAM17 in hepatocytes leads to lower levels of EGFR activating factors in the serum from peripheral blood after 2/3 partial hepatectomy. This was demonstrated by lower levels of phosphorylated AKT in ADAM10ΔAlbADAM17ΔAlb mice 6 hours post hepatectomy, which was not observed in single deficient mice. Moreover, decreased phosphorylation of EGFR was observed in the cultured cells treated by mouse serum originating from ADAM10ΔAlbADAM17ΔAlb mice after hepatectomy. Altogether this indicates that hepatocytes alone are a significant source of growth factors which modulate their cell cycle. Similar results were described by McMahan et al. [33] in hepatocyte-specific ADAM17-deficient mice, in which dramatically reduced levels of AREG were observed. However, AREG levels in their study were measured in liver tissue lysates and therefore represented the expression of a protein, not its shed pool. In our model we did not observe differences of expression in AREG protein but we clearly showed that the deletion of ADAM proteases influenced release of EGFR ligands to the serum. Our data indicate that not only ADAM17, but also ADAM10 is a regulator of EGFR ligand shedding after partial hepatectomy. Of note is that factors released from hepatocytes can potentially influence also other liver cell types, e.g. hepatic stellate cells. This may have a negative impact in conditions such as liver fibrosis when enhanced HSC proliferation is not desired.
Similarly to EGFR, HGF signalling through c-Met receptor is another independent pathway leading to the proliferation of hepatocytes [7] [40]. In our model, serum HGF was strongly increased after partial hepatectomy. On the other hand, we showed that shedding of c-Met was also elevated, especially in the first hours after the hepatectomy, which is probably part of a regulatory mechanism of HGF signalling. We observed that isolated primary ADAM10 and ADAM17 double-deficient hepatocytes had a more prominent response to HGF treatment. We concluded that these differences in HGF response were caused by higher availability of c-Met receptor on cell surface due to its limited shedding in the combined absence of ADAM10 and ADAM17. Shedding of c-Met did not significantly differ in the absence of ADAM10 or ADAM17 individually. The question remains whether shedding of the c-Met receptor results only in lower availability of functional receptor on the cell surface, or if there is an additional contribution of a solubilized part of the receptor. Released cleft fragment of c-Met could bind HGF and prevent binding of HGF to functional surface-bound receptors. We did not identify this decoy function of a c-Met fragment in our in vitro conditions, but further investigation is desired.
In summary, the deletion of ADAM10 and ADAM17 driven by the albumin promoter leads to hampered EGFR signalling, but enhanced HGF/c-Met signalling in regenerating hepatocytes. As a result, ADAM10ΔAlbADAM17ΔAlb mice did not show any alteration from Ctrl littermates 40 hours post hepatectomy, hepatocytes proceeded towards proliferation and liver weights increased normally.
We have reported previously that liver-specific ADAM10-deficient mice, with stronger expression of Cre (Alb-Cre homozygotes or α-fetoprotein-Cre hemizygotes), developed spontaneous fibrosis [23]. This was not the case in mice used in this study (Alb-Cre hemizygotes). ADAM10ΔAlb mice were monitored up to 33 weeks of age, and none of the fibrotic markers were increased in unchallenged animals. The lower expression levels of Cre in hemizygote animals resulted in sustained expression of ADAM10 in a negligible proportion of hepatocytes, which was sufficient to protect from development of spontaneous fibrosis. However, ADAM10ΔAlb mice were much more susceptible to liver damage than control littermates when exposed to the toxic agent. Difference was not apparent after acute intoxication (single dose of CCl4) but was clearly distinct after 4 weeks of chronic CCl4 treatment. The reason for the higher susceptibility of ADAM10-deficient mice towards CCl4 intoxication is still not clear. It was shown that ADAM10 directly influences the expression of bile acid transporters, e.g. MRP2 [23], which play an important role in removal of toxic products generated in hepatocytes after CCl4 treatment. However, we did not find the difference in Mrp2 mRNA expression, nor in the expression of Mrp3 and Mrp4 which are involved in CCl4 detoxication [41]. The difference in Mrp2 expression between two studies may have arisen due to the differing nature of the injuries evoked in the studies, as we used toxin- induced injury, while model of Muller et al. [23] resulted from an inner imbalance of liver homeostasis.
Not only levels of ALT and AST were increased in ADAM10ΔAlb, but also ALP. ALP in liver damage is connected with activated biliary epithelial cell. This is in line with previously published data which suggest that ADAM10-deficient mice have increased ductular reaction [23]. Consistently with this, we found that ADAM10ΔAlb had increased protein levels of CD44 in liver lysates. Biliary epithelial cell are a prominent source of CD44 in liver [42], and its expression is connected to the proliferation of biliary epithelia. The identified increase of CD44 levels could be directly linked to ADAM10 deficiency, as ADAM10 is the primary sheddase for CD44 [43].
Interestingly, the hepatocyte damage in ADAM10ΔAlbADAM17ΔAlb and ADAM17ΔAlb mice was comparable to Ctrl littermates. Thus, ADAM17 elimination was not protective in CCl4 induced liver fibrosis when functional ADAM10 was present, but alleviated aggravating impact of ADAM10 deficiency on fibrosis development. Similarly, we have shown previously [44] that ADAM17 inhibition by ursodeoxycholic acid is protective in model of cholestatic injury. In contrast, recent work from Sundaram et al. [33], showed that impaired ADAM17 maturation exacerbated bile duct obstruction induced fibrosis. The main difference between their work and our present findings is the cell type affected by genetic models. Our model leads to specific ablation of ADAM17 in hepatocytes and biliary epithelial cells. Sundaram et al. studied mice with whole body deficiency of iRhom2 that leads to the inhibition of ADAM17 in cell types, in which iRhom2 governs maturation of ADAM17. They explained their phenotype through the suppression of ADAM17 in hepatic stellate cells, a cell type which was not affected in our genetic model. This shows that the modulation of ADAM17 activity on different cell types within the liver can lead to opposite pathological consequences.
In summary, ADAM10ΔAlb are more prone to chronic intoxication, but subsequent proliferation of hepatocytes is not affected. Interestingly, ADAM17 deficiency counteracted the negative effect of ADAM10 deficiency in liver fibrosis development. ADAM10ΔAlbADAM17ΔAlb animals were the same as Ctrl littermates in all studied parameters during fibrosis development. Further deciphering of molecular mechanism behind this effect represent interesting area for future research.
Role of ADAM17 in TNF-α shedding is very well-known. However, growing number of evidence suggest the importance of TNF receptors shedding in the regulation of TNF-α signalling [32]. Shedding of TNF RI or TNF RII not only disables functionality of this receptor on the cell surface but released part - soluble receptor, can bind and limit the availability of TNF- α. Role of TNF RI in liver injury is not easy to interpret. Inhibition of TNF RI led to lower hepatocellular damage after CCl4 treatment [45]. On the other hand, the different report described the importance of TNF RI or liver regeneration after CCl4 intoxication, as mice lacking TNF RI showed inhibited hepatocyte DNA synthesis [46]. A recent study showed that TNF RI deficiency led to the increased cholestatic injury in the model of chronic hepatitis, mediated through higher infiltration of immune cells [47]. In our study, we found that ADAM17 deficiency in hepatocytes leads to lower levels of TNFRI in serum in both studied liver challenges, as expected. Surprisingly, ADAM10-deficient animals consistently show higher levels of TNF RI in serum than their Ctrl littermates. Same results were confirmed in vitro on primary hepatocytes, suggesting elevated serum levels of sTNF RI in vivo had a source in ADAM10-deficient hepatocytes. This effect is most likely not caused by compensational activation of ADAM17, as other substrates of ADAM17, TNF-α and AREG, were not elevated in the medium of ADAM10-deficient cells. Moreover, ADAM10ΔAlbADAM17ΔAlb mice show an interesting pattern in levels of TNF RI in serum. In an unchallenged state, ADAM10ΔAlbADAM17ΔAlb exhibit increased shedding of TNF RI similarly to ADAM10ΔAlb, while during the liver challenge, double deficient mice were comparable to ADAM17ΔAlb or Ctrl littermates. This nicely shows that the activity of ADAM17 is induced in liver pathological states and the lack of this induction becomes evident in ADAM10ΔAlbADAM17ΔAlb after the challenge, while unchallenged animals resemble more ADAM10ΔAlb in respect to TNF RI shedding. Besides proteolytic shedding, soluble TNF RI can be released into serum by exosome-like vesicles [48]. It would be worth to investigate whether ADAM10 deficiency alters this export.
Altogether our data show, that hepatocyte-derived ADAM10 and ADAM17 contribute to the regulation of several distinct pathways, i.e. EGFR, HGF and TNF RI, during liver injury and regeneration. Potential therapies based on inhibition of ADAM10 and ADAM17 should carefully consider various roles of these proteases in different cell types.