Dynein Light Chain LC8 Negatively Regulates NF-κB through the Redox-dependent Interaction with IκBα

Abstract

Redox regulation of nuclear factor B (NF-B) has been described, but the molecular mechanism underlying such regulation has remained unclear. We recently showed that a novel disulfide reductase, TRP14, inhibits tumor necrosis factor ␣ (TNF␣)-induced NF-B activation, and we identified the dynein light chain LC8, which interacts with the NF-B inhibitor IB␣, as a potential substrate of TRP14. We now show the molecular mechanism by which NF-B activation is redox-dependently regulated through LC8. LC8 inhibited TNF␣-induced NF-B activation in HeLa cells by interacting with IB␣ and thereby preventing its phosphorylation by IB kinase (IKK), without affecting the activity of IKK itself. TNF␣ induced the production of reactive oxygen species, which oxidized LC8 to a homodimer linked by the reversible formation of a disulfide bond between the Cys 2 residues of each subunit and thereby resulted in its dissociation from IB␣. Butylated hydroxyanisol, an antioxidant, and diphenyleneiodonium, an inhibitor of NADPH oxidase, attenuated the phosphorylation and degradation of IB␣ by TNF␣ stimulation. In addition LC8 inhibited NF-B activation by other stimuli including interleukin-1␤ and lipopolysaccharide, both of which generated reactive oxygen species. Furthermore, TRP14 catalyzed reduction of oxidized LC8. Together, our results indicate that LC8 binds IB␣ in a redoxdependent manner and thereby prevents its phosphorylation by IKK. TRP14 contributes to this inhibitory activity by maintaining LC8 in a reduced state.

Dynein Light Chain LC8 Inhibits Osteoclast Differentiation and Prevents Bone Loss in Mice

“…In response to potent NF-kB activators such as TNF-a, IkBa is phosphorylated at residues Ser 32 and Ser 36 by IKK and subsequently degraded by the ubiquitin-proteasome system (33). Pre- vious studies showed that LC8 blocked IkBa phosphorylation by IKK in TNF-a-treated cells (25). Therefore, we examined the effect of LC8 on IkBa phosphorylation and degradation in BMMs stimulated with RANKL.…”

“…The DNA sequence encoding hemagglutinin (HA)-tagged LC8 was amplified via PCR using the pCGN-LC8 vector (25,26) as a template with a forward primer (59-CG GAA TTC ATG GCT TCT AGC TAT CCT TAT GAC-39) containing both an EcoRI site (underlined) and the initiation codon (bold) and a reverse primer (59-GG GGT ACC TTA ACC AGA TTT GAA CAG AAG AAT G-39) containing both a KpnI site (underlined) and the stop codon (bold). The PCR product was purified, digested with restriction enzymes EcoRI and KpnI, and cloned into the corresponding sites of the pCBA-M vector.…”

“…In a previous study, we identified LC8 as a potential substrate of 14-kDa human thioredoxin-related protein (TRP14), a disulfide reductase that inhibits TNF-a-induced NF-kB activation by suppressing the phosphorylation of IkBa (24). More recently, we showed that LC8 inhibits IkBa phosphorylation by IkB kinase (IKK) via its redoxdependent interaction with IkBa and that TRP14 regulates this inhibitory activity by facilitating the reduction of LC8 (25,26).…”

“…In addition, dominant-negative IkBa proteins (29) and NF-kB inhibitors (30) were shown to block osteoclastogenesis. Moreover, previous studies have shown that LC8 inhibits NF-kB activation by potent NF-kB stimulators, including TNF-a, LPS, and IL-1b (25,26). Therefore, we sought to determine whether LC8 inhibits RANKL-induced NF-kB activation and subsequent osteoclast differentiation and bone resorption using transgenic (Tg) mice with ectopic expression of LC8.…”

Basic Principles and Emerging Concepts in the Redox Control of Transcription Factors

“…By maintaining LC8 in the reduced state, TRP14 prevents cytosolic activation of NF-jB. Exposure to oxidants or treatment with TNFa, IL-1, or LPS known to signal via H 2 O 2 production leads to the formation of LC8 dimers in which two molecules are linked via a disulfide bridge (515), resulting in its dissociation from IjB (224,227). This mode of redox-dependent inhibition of protein activities by interaction with binding partners is analogous to the association of Trx to ASK-1, an upstream activator of the c-Jun N-terminal kinase (INK) and p-38 MAPK signaling pathways (415) (Fig.…”