Simulated Microgravity Inhibits Rodent Dermal Fibroblastic Differentiation of Mesenchymal Stem Cells by Suppressing ERK/β-Catenin Signaling Pathway

Studies have shown that bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into dermal fibroblasts to participate in skin-repairing. However, at present, little is known about how microgravity affects dermal fibroblastic differentiation of BMSCs in space. The aim of this study was to investigate the effect of simulated microgravity (SMG) on the differentiation of BMSCs into dermal fibroblasts and the related molecular mechanism. Here, using a 2D-clinostat device to simulate microgravity, we found that SMG inhibited the differentiation and suppressed the Wnt/β-catenin signaling and phosphorylation of extracellular regulated protein kinases 1/2 (ERK1/2). After upregulating the Wnt/β-catenin signaling with lithium chloride (LiCl) treatment, we found that the effect of the differentiation was restored. Moreover, the Wnt/β-catenin signaling was upregulated when phosphorylation of ERK1/2 was activated with tert-Butylhydroquinone (tBHQ) treatment. Taken together, our findings suggest that SMG inhibits dermal fibroblastic differentiation of BMSCs by suppressing ERK/β-catenin signaling pathway, inferring that ERK/β-catenin signaling pathway may act as a potential intervention target for repairing skin injury under microgravity conditions.

GSK3β rephosphorylation rescues ALPL deficiency-induced impairment of odontoblastic differentiation of DPSCs

Background Premature exfoliation of the deciduous teeth is a common manifestation in childhood patients with hypophosphatasia (HPP), which is an autosomal inherited disease caused by ALPL mutations. Dysplasia of the cementum, dentin, and alveolar bone has been proposed to be the main reasons for the exfoliation of teeth, while the extraordinarily complex intracellular mechanisms remain elusive. Dental pulp stem cells (DPSCs) have been demonstrated to successfully regenerate functional pulp-dentin-like tissue. Dental pulp cells derived from HPP patients impaired mineralization; however, insight into the deeper mechanism is still unclear. Methods The effects of ALPL on odontoblastic differentiation of DPSCs from HPP patient were assessed by Alizarin Red staining, immunofluorescent staining, Western blot and RT-PCR, and micro-CT assays. Result Here, we found DPSCs from HPP patient exhibited low ALP activity and impaired odontoblastic differentiation. Meanwhile, we found that loss of function of ALPL reduced phosphorylation of GSK3β in DPSCs. While GSK3β rephosphorylation improved odontoblastic differentiation of HPP DPSCs with LiCl treatment. Finally, we demonstrated systemic LiCl injection ameliorated tooth-associated defects in ALPL+/− mice by enhanced phosphorylation of GSK3β in the teeth. Conclusions Our study indicates that ALPL regulates odontoblastic differentiation of DPSCs and provides useful information for understanding how ALPL deficiency led to tooth dysplasia and, ultimately, may inform efforts at improvement tooth defects in HPP patients.

Lithium Reduces Migration and Collagen Synthesis Activity in Human Cardiac Fibroblasts by Inhibiting Store-Operated Ca2+ Entry

Cardiac fibrosis plays a vital role in the pathogenesis of heart failure. Fibroblast activity is enhanced by increases in store-operated Ca2+ entry (SOCE) and calcium release-activated calcium channel protein 1 (Orai1) levels. Lithium regulates SOCE; however, whether therapeutic concentrations of lithium can be used to inhibit cardiac fibrogenesis is unknown. Migration and proliferation assays, Western blotting, real-time reverse-transcription polymerase chain reaction analysis, and calcium fluorescence imaging were performed in human cardiac fibroblasts treated with or without LiCl at 1.0 mM (i.e., therapeutic peak level) or 0.1 mM (i.e., therapeutic trough level) for 24 h. Results showed that LiCl (0.1 mM, but not 1.0 mM) inhibited the migration and collagen synthesis ability of cardiac fibroblasts. Additionally, thapsigargin-induced SOCE was reduced in fibroblasts treated with LiCl (0.1 mM). The expression level of Orai1 was lower in LiCl (0.1 mM)-treated fibroblasts relative to the fibroblasts without LiCl treatment. Fibroblasts treated with a combination of LiCl (0.1 mM) and 2-APB (10 μM, an Orai1 inhibitor) demonstrated similar migration and collagen synthesis abilities as those in LiCl (0.1 mM)-treated fibroblasts. Altogether, lithium at therapeutic trough levels reduced the migration and collagen synthesis abilities of human cardiac fibroblasts by inhibiting SOCE and Orai1 expression.

Effects of chronic lithium treatment on anxious behaviors and serotonergic genes expression in the midbrain raphe nuclei in defeated male mice

There are experimental data that mixed anxiety/depression-like state induced by chronic social defeat stress is accompanied by development of anxiety and downregulation of serotonergic gene expression in the midbrain raphe nuclei of male mice. The paper aimed to study the effect of chronic lithium chloride (LiCl) on anxious behaviors and the expression of serotonergic genes (Tph2, Slc6a4, Htr1a, Htr5b) in the midbrain raphe nuclei of defeated mice. Slight anxiolytic effects of LiCl were found on the commucativeness in the partition test, and anxiogenic-like effects, estimated by the elevated plus-maze and social interactions tests. Chronic LiCl treatment induced overexpression of the serotonergic genes in the midbrain raphe nuclei of defeated mice. We can assume that effects of LiCl, rather anxiogenic, may be due to activation of serotonergic system induced by hyperexpression of serotonergic genes. Our findings will allow to understand the factors involved in the positive and side effects of lithium on anxiety and function of serotonergic genes which are involved into mechanisms of depression.

GSK3β Rephosphorylation Rescues ALPL Deficiency Induced-impairment of Odontoblastic Differentiation of DPSCs

Background: Premature exfoliation of deciduous teeth is a common manifestation in childhood patients with Hypophosphatasia (HPP), which is an autosomal inherited disease caused by ALPL mutations. Dysplasia of cementum, dentin and alveolar bone have been proposed to be the main reasons for the exfoliation of teeth, while the extraordinarily complex intracellular mechanisms remain elusive. Dental pulp stem cells (DPSCs) have been demonstrated to successfully regenerate functional pulp-dentin like tissue. Dental pulp cells derived from HPP patients impaired mineralization, however insight into the deeper mechanism is still unclear. Methods: The effects of ALPL on odontoblastic differentiation of DPSCs from HPP patient were assessed by Alizarin Red staining, immunofluorescent staining, Western blot and RT-PCR, and micro-CT assays. Results: Here, we found DPSCs from HPP patient exhibited low ALP activity and impaired odontoblastic differentiation. Meanwhile, we found that loss of function of ALPL reduced phosphorylation of GSK3β in DPSCs. While GSK3β rephosphorylation improved odontoblastic differentiation of HPP DPSCs with LiCl treatment. Finally, we demonstrated systemic LiCl injection ameliorated tooth associated defects in ALPL+/- mice by enhanced phosphorylation of GSK3β in teeth. Conclusions: Our study indicates that ALPL regulates odontoblastic differentiation of DPSCs, and provides useful information for understanding how ALPL deficiency leaded to tooth dysplasia and, ultimately, may inform efforts at improvement tooth defects in HPP patients.

Multi-level regulation of PKCδI alternative splicing by lithium chloride

Lithium chloride (LiCl) is commonly used in treatment of mood disorders, however its usage leads to weight gain which promotes metabolic disorders. Protein Kinase C delta (PKCδ), a serine/threonine kinase, is alternatively spliced to PKCδI and PKCδII in 3T3L1 cells. We previously demonstrated that PKCδI is the predominantly expressed isoform in 3T3L1 pre-adipocytes. Here, we demonstrate that LiCl treatment decreases PKCδI levels, increases formation of lipid droplets and increases oxidative stress. Hence, we investigated the molecular mechanisms underlying the regulation of PKCδI alternative splicing by LiCl. We previously demonstrated that the splice factor SFRS10 is essential for PKCδI splicing. Our results demonstrate that GSK3ß phosphorylates SFRS10 and SFRS10 is in a complex with long noncoding RNA NEAT1 to promote PKCδI splicing. Using PKCδ splicing minigene and RNA-immunoprecipitation assays, our results demonstrate that upon LiCl treatment, NEAT1 levels are reduced, GSK3ß activity is inhibited and SFRS10 phosphorylation is decreased which leads to decreased expression of PKCδI. Integration of the GSK3ß signaling pathway with the ribonucleoprotein complex of lncRNA NEAT1 and SFRS10 enables fine tuning of PKCδI expression during adipogenesis. Knowledge of the molecular pathways impacted by LiCl provide an understanding of the ascent of obesity as a comorbidity in disease management.

Hyperosmolar Ionic Solutions Modulate Inflammatory Phenotype and sGAG Loss in a Cartilage Explant Model

Objective The objective of this study was to compare the effects of hyperosmolar sodium (Na+), lithium (Li+) and potassium (K+) on catabolic and inflammatory osteoarthritis (OA) markers and sulfated glycosaminoglycan (sGAG) loss in TNF-α-stimulated cartilage explants. Methods Explants from bovine stifle joints were stimulated with TNF-α for 1 day to induce cartilage degradation followed by supplementation with 50 mM potassium chloride (KCl), 50 mM lithium chloride (LiCl), 50 mM sodium chloride (NaCl), or 100 nM dexamethasone for an additional 6 days. We assessed the effect of TNF-α stimulation and hyperosmolar ionic treatment on sGAG loss and expression of OA-associated proteins: ADAMTS-5, COX-2, MMP-1, MMP-13, and VEGF. Results TNF-α treatment increased sGAG loss ( P < 0.001) and expression of COX-2 ( P = 0.018), MMP-13 ( P < 0.001), and VEGF ( P = 0.017) relative to unstimulated controls. Relative to activated controls, LiCl and dexamethasone treatment attenuated sGAG loss ( P = 0.008 and P = 0.042, respectively) and expression of MMP-13 ( P = 0.005 and P = 0.036, respectively). In contrast, KCl treatment exacerbated sGAG loss ( P = 0.032) and MMP-1 protein expression ( P = 0.010). NaCl treatment, however, did not alter sGAG loss or expression of OA-related proteins. Comparing LiCl and KCl treatment shows a potent reduction ( P < 0.05) in catabolic and inflammatory mediators following LiCl treatment. Conclusion These results suggest that these ionic species elicit varying responses in TNF-α-stimulated explants. Cumulatively, these findings support additional studies of hyperosmolar ionic solutions for potential development of novel intraarticular injections targeting OA.

GANT61 and Lithium Chloride Inhibit the Growth of Head and Neck Cancer Cell Lines Through the Regulation of GLI3 Processing by GSK3β

Several signaling pathways are aberrantly activated in head and neck squamous cell carcinoma (HNSCC), including the Hedgehog-Gli (HH-GLI), WNT, EGFR, and NOTCH pathways. The HH-GLI pathway has mostly been investigated in the context of canonical signal transduction and the inhibition of the membrane components of the pathway. In this work we investigated the role of downstream inhibitors GANT61 and lithium chloride (LiCl) on cell viability, wound closure, and colony forming ability of HNSCC cell lines. Five HNSCC cell lines were treated with HH-GLI pathway inhibitors affecting different levels of signal transduction. GANT61 and LiCl reduce the proliferation and colony formation capabilities of HNSCC cell lines, and LiCl has an additional effect on wound closure. The major effector of the HH-GLI signaling pathway in HNSCC is the GLI3 protein, which is expressed in its full-length form and is functionally regulated by GSK3β. LiCl treatment increases the inhibitory Ser9 phosphorylation of the GSK3β protein, leading to increased processing of GLI3 from full-length to repressor form, thus inhibiting HH-GLI pathway activity. Therefore, downstream inhibition of HH-GLI signaling may be a promising therapeutic strategy for HNSCC.

Lithium Chloride Sensitivity in Yeast and Regulation of Translation

For decades, lithium chloride (LiCl) has been used as a treatment option for those living with bipolar disorder (BD). As a result, many studies have been conducted to examine its mode of action, toxicity, and downstream cellular responses. We know that LiCl is able to affect cell signaling and signaling transduction pathways through protein kinase C and glycogen synthase kinase-3, which are considered to be important in regulating gene expression at the translational level. However, additional downstream effects require further investigation, especially in translation pathway. In yeast, LiCl treatment affects the expression, and thus the activity, of PGM2, a phosphoglucomutase involved in sugar metabolism. Inhibition of PGM2 leads to the accumulation of intermediate metabolites of galactose metabolism causing cell toxicity. However, it is not fully understood how LiCl affects gene expression in this matter. In this study, we identified three genes, NAM7, PUS2, and RPL27B, which increase yeast LiCl sensitivity when deleted. We further demonstrate that NAM7, PUS2, and RPL27B influence translation and exert their activity through the 5′-Untranslated region (5′-UTR) of PGM2 mRNA in yeast.

G-Alpha Subunit Abundance and Activity Differentially Regulate β-Catenin Signaling

ABSTRACT Heterotrimeric G proteins are signal transduction proteins involved in regulating numerous signaling events. In particular, previous studies have demonstrated a role for G-proteins in regulating β-catenin signaling. However, the link between G-proteins and β-catenin signaling is controversial and appears to depend on G-protein specificity. We describe a detailed analysis of a link between specific G-alpha subunits and β-catenin using G-alpha subunit genetic knockout and knockdown approaches. The Pasteurella multocida toxin was utilized as a unique tool to activate G-proteins, with LiCl treatment serving as a β-catenin signaling agonist. The results show that Pasteurella multocida toxin (PMT) significantly enhanced LiCl-induced active β-catenin levels in HEK293T cells and mouse embryo fibroblasts. Evaluation of the effect of specific G-alpha proteins on the regulation of β-catenin showed that Gq/11 and G12/13 knockout cells had significantly higher levels of active and total β-catenin than wild-type cells. The stimulation of active β-catenin by PMT and LiCl was lost upon both constitutive and transient knockdown of G12 and G13 but not Gq. Based on our results, we conclude that endogenous G-alpha proteins are negative regulators of active β-catenin; however, PMT-activated G-alpha subunits positively regulate LiCl-induced β-catenin expression in a G12/13-dependent manner. Hence, G-alpha subunit regulation of β-catenin is context dependent.