p38α phosphorylation was increased in WT BDL mice upon chronic cholestasis (Fig. 2). This activation of p38α led to a significant increase in MAPK-activated kinase 2 (MK2) phosphorylation on threonine 334. Indeed, only in WT BDL mice there was a significant increase in phosphorylation of MK2 and, therefore, activation of MK2, when compared with WT sham mice and KO mice. It has been reported that
MK2 can phosphorylate Akt on serine 473.14 We also tested two other regulators of Akt, phosphoinositide-dependent kinase-1 (PDK1) and phosphatase and tensin homolog (PTEN), but no differences were found in their phosphorylation and protein levels among groups. Similar results were obtained 12 days after BDL (data not shown). Quantification of the western blots is shown in Supporting Fig. S3. The p38α downstream pathway was MK0683 in vivo assessed starting with one of its major targets, MK2. As shown in Fig. 2B, phosphorylation of MK2 on threonine 334 was strongly regulated by this pathway. However, neither PDK-1 nor PTEN levels and phosphorylation were modified upon p38α deficiency. Akt may be phosphorylated on serine
473 by p-MK2 and this phosphorylation was markedly reduced upon p38 deficiency, whereas phosphorylation on threonine 308 remained unaffected (Fig. 3A). Other downstream targets such as mammalian target of rapamycin (mTOR) and glycogen synthase kinase (GSK) 3β were phosphorylated after BDL in a p38α-dependent manner (Fig. 3B). GSK3β phosphorylation only increased markedly in WT BDL mice, which would inactivate the enzyme. One of the major targets of GSK3β is β-catenin, which exhibited Protein Tyrosine Kinase inhibitor an increase only in BDL WT mice. The same western blots
were performed with mice after 12 days of BDL (results not shown). The inflammatory and profibrogenic profiles were assessed in WT and p38α KO mice (Fig. 4). p38α KO mice had higher messenger RNA (mRNA) levels of some proinflammatory cytokines, such as RANTES under basal conditions (Fig. 4B), and the BDL group had higher mRNA levels of adhesion factor Icam-1 (Fig. 4C). Although TNF-α expression was not affected by the absence of p38α, the mRNA levels of receptor 1 for TNF-α increased in p38α KO mice, making these animals MCE公司 likely more sensitive to this cytokine (Fig. 4A). On the other hand, the antiinflammatory cytokine IL-10 mRNA level markedly increased in p38α KO BDL mice after 12 days of BDL (Fig. 4B), probably to restrain the inflammatory response. STAT3 phosphorylation was increased after BDL similarly in both WT and KO mice (Supporting Fig. S4). However, no significant changes in phosphorylation of p65 were found upon BDL (Supporting Fig. S4). Liver-specific p38α-deficient mice did not show a higher degree of apoptosis upon chronic cholestasis compared with WT mice (Fig. S5). Indeed, the cleavage of caspase 3 (Fig. S5) showed no further increase in apoptosis upon p38α deficiency.