Redundant roles for cJun-N-terminal kinase 1 and 2 in interleukin-1β-mediated reduction and modification of murine hepatic nuclear retinoid X receptor α

Background/Aims

Retinoid X receptor α (RXRα), the heterodimeric partner for multiple nuclear receptors (NRs), was shown to be an essential target for inflammation-induced cJun-N-terminal kinase (JNK) signaling in vitro. This study aimed to explore the role of hepatic JNK signaling and its effects on nuclear RXRα levels downstream of interleukin-1β (IL-1β) in vivo.

Methods

Effects of IL-1β on hepatic NR-dependent gene expression, nuclear RXRα levels, and roles for individual JNK isoforms were studied in wild-type, Jnk1^−/−, and Jnk2^−/− mice and in primary hepatocytes of each genotype.

Results

IL-1β administration showed a time-dependent reduction in expression of the hepatic NR-dependent genes Ntcp, Cyp7a1, Cyp8b1, Abcg5, Mrp2, and Mrp3. IL-1β treatment for 1 h activated JNK and resulted in both post-translational modification and reduction of nuclear RXRα. In wild-type primary hepatocytes, IL-1β modified and reduced nuclear RXRα levels time dependently, which was prevented by chemical inhibition of JNK as well as by inhibition of proteasomal degradation. Individual absence of either JNK1 or JNK2 did not significantly influence the reduction or modification of hepatic nuclear RXRα by IL-1β both in vivo and in primary hepatocytes.

Conclusions

Functional redundancy exists for JNK1 and JNK2 in IL-1β-mediated alterations of hepatic nuclear RXRα levels, stressing the importance of this pathway in mediating the hepatic response to inflammation.

Introduction

Bacterial products, including lipopolysaccharide (LPS) released by gram-negative bacteria, are primary inducers of the physiologic manifestations of sepsis. By binding to its cognate receptor toll-like receptor-4 (TLR-4), LPS causes the release of inflammatory cytokines from nonparenchymal cells [1], [2], [3], which activate intracellular signaling pathways in hepatocytes, thereby inducing broad changes in hepatic gene expression (i.e., the acute phase response [APR]). LPS can also directly activate TLR-4 that is present on hepatocytes [4], [5]. In hepatocytes, the negative APR involves downregulation of expression of key transport proteins regulating uptake and secretion of most biliary components, including reduced sinusoidal uptake and canalicular excretion of bile acids by suppression of Na⁺/taurocholate cotransporting polypeptide (Ntcp; solute carrier family 10 [sodium/bile acid cotransporter family], member 1 [Slc10a1]) and Abcb11 (ATP-binding cassette, sub-family B (MDR/TAP), member 11), respectively, as well as reduced expression of multiple canalicular transporters including the polyspecific organic cation transporter Mrp2 (Abcc2), the heterodimeric cholesterol transporters Abcg5/g8, and the phospholipid transporter Mdr2 (Abcb4) [6]. Together, this subsequently leads to a reduction in bile flow; accumulation of toxic compounds, including bile acids in liver and serum; and, eventually, liver damage. Many of these genes are regulated by type 2 nuclear receptors (NRs), which are ligand-activated transcription factors that require heterodimerization with retinoid X receptor α (RXRα; nuclear receptor sub-family 2, group B, member 1 [NR2B1]) to fully function. Previous studies in rodents [6], [7], [8], [9], [10], [11], [12], [13], [14] indicated that LPS reduced the expression of NR-dependent genes due to decreased binding of regulatory nuclear proteins to their DNA-binding elements, including type 2 NRs. Recent studies from our laboratory have demonstrated reduced hepatic nuclear RXRα protein levels after LPS administration in vivo and IL-1β in vitro, and as a common partner of multiple NRs, this appears to be a major contributor of reduced hepatic gene expression during negative hepatic APR [6], [9], [15]. Studies in HepG2 cells [15] support a mechanism in which IL-1β-induced signaling resulted in phosphorylation of nuclear RXRα at serine 260, which required activation of c-Jun-N-terminal kinase (JNK) and induced export of the majority of nuclear RXRα to the cytosol for degradation by the proteasome. From these and other studies, it is clear that JNK-dependent pathways are centrally involved in hepatic inflammatory responses [2], [16], [17], [18].

The liver expresses two JNK genes, Jnk1 and Jnk2, each consisting of two alternative splicing forms, p54 and p46 [19], [20], [21]. Individual Jnk1 and Jnk2 knockout mice are viable [20], [21], whereas the Jnk1/Jnk2 double knockout is not [22]. Several recent studies have demonstrated shared and distinct functions for JNK1 and JNK2 [23], [24], [25], [26], [27]. In primary hepatocytes, for example, deoxycholic acid-induced toxicity is mediated via JNK1, whereas JNK2 is protective [26]. Additionally, JNK1 and JNK2 play opposite roles in the development of type 2 and type 1 diabetes, respectively [24], [28], in Th1 and Th2 inflammatory responses [21], [29], [30], [31] and in obesity and hepatic steatohepatitis [32]. It is not known if JNK isoforms play distinct roles in the negative hepatic APR or specifically in mediating IL-1β changes in RXRα function. Given the central role for IL-1β in the hepatic APR [33], [34], [35] and the essential roles for RXRα in a wide variety of hepatic functions [2], [16], [36], [37], we aimed to specifically explore roles for JNK1 and JNK2 in the response of the liver to IL-1β, with a focus on nuclear RXRα levels and function.