P21 (Cdc42/Rac)-activated kinase 1 (pak1) is associated with cardiotoxicity induced by antihistamines.

Astemizole, a non-sedating histamine H1 receptor blocker, is widely known to cause cardiac arrhythmia, which prolongs the QT interval. However, the precise molecular mechanism involved in antihistamine-induced cardiovascular adverse effects other than hERG channel inhibition is still unclear. In this study, we used DNA microarray analysis to detect the mechanisms involved in life-threatening adverse effects caused by astemizole. Rat primary cardiomyocytes were treated with various concentrations of astemizole for 24 h and the corresponding cell lysates were analyzed using a DNA microarray. Astemizole altered the expression profiles of genes involved in calcium transport/signaling. Using qRT-PCR analysis, we demonstrated that, among those genes, p21 (Cdc42/Rac)-activated kinase 1 (pak1) mRNA was downregulated by treatment with terfenadine and astemizole. Astemizole also reduced pak1 protein levels in rat cardiomyocytes. In addition, astemizole decreased pak1 mRNA and protein levels in H9c2 cells and induced an increase in cell surface area (hypertrophy) and cytotoxicity. Fingolimod hydrochloride (FTY720), a pak1 activator, inhibited astemizole-induced hypertrophy and cytotoxicity in H9c2 cells. These results suggest that antihistamine-induced cardiac adverse effects are associated with pak1 expression and function.

Nrf2 deficiency induces oxidative stress and promotes RANKL-induced osteoclast differentiation.

Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates the expression of a variety of antioxidant and detoxification genes through an antioxidant-response element. Nrf2 has been shown to protect several types of cells against the acute and chronic injury that accompanies oxidative stress, but its role in osteoclasts remains unclear. In this study, we investigated the role of Nrf2 in osteoclast (OC) differentiation, a process in which reactive oxygen species (ROS) are generated and then participate, using Nrf2-knockout mice. Receptor activator of nuclear factor κB ligand (RANKL)-induced OC differentiation, actin ring formation, and osteoclastic bone resorption were substantially promoted in Nrf2-deficient OC precursor cells compared to wild-type cells. Under both unstimulated and RANKL-stimulated conditions, Nrf2 loss led to an increase in the intracellular ROS level and the oxidized-to-reduced glutathione ratio and a defect in the production of numerous antioxidant enzymes and glutathione. Moreover, pretreatment with N-acetylcysteine or diphenyleneiodonium significantly reduced the OC differentiation and decreased the intracellular ROS level in both Nrf2-deficient and wild-type cells. Pretreatment with sulforaphane and curcumin also inhibited the OC differentiation by activating Nrf2 in part. Nrf2 deficiency promoted the RANKL-induced activation of mitogen-activated protein kinases, including c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38; the induction of c-Fos; and the consequent induction of nuclear factor of activated T cells, cytoplasmic 1, a pivotal determinant of OC differentiation. Our results suggest that Nrf2 probably inhibits RANKL-induced OC differentiation by regulating the cellular redox status by controlling the expression of oxidative response genes, findings that might form the basis of a new strategy for treating inflammatory bone diseases.

Dynein light chain LC8 inhibits osteoclast differentiation and prevents bone loss in mice.

NF-κB is one of the key transcription factors activated by receptor activator of NF-κB ligand (RANKL) during osteoclast differentiation. The 8-kDa dynein L chain (LC8) was previously identified as a novel NF-κB regulator. However, its physiological role as an NF-κB inhibitor remains elusive. In this study, we showed the inhibitory role of LC8 in RANKL-induced osteoclastogenesis and signaling pathways and its protective role in osteolytic animal models. LC8 suppressed RANKL-induced osteoclast differentiation, actin ring formation, and osteoclastic bone resorption. LC8 inhibited RANKL-induced phosphorylation and subsequent degradation of IκBα, the expression of c-Fos, and the consequent activation of NFATc1, which is a pivotal determinant of osteoclastogenesis. LC8 also inhibited RANKL-induced activation of JNK and ERK. LC8-transgenic mice exhibited a mild osteopetrotic phenotype. Moreover, LC8 inhibited inflammation-induced bone erosion and protected against ovariectomy-induced bone loss in mice. Thus, our results suggest that LC8 inhibits osteoclast differentiation by regulating NF-κB and MAPK pathways and provide the molecular basis of a new strategy for treating osteoporosis and other bone diseases.