TSH activates macrophage inflammation by G13 and G15-dependent pathways

Thyroid-stimulating hormone (TSH) treatment activates IκB/NFκB (p65) and ERK, P38 in macrophages, but how these pathways are activated, and how they contribute to the pro-inflammatory effect of TSH on macrophages remain unknown. TSH receptor (TSHR) is coupled to four subfamilies of G proteins (Gs, Gi/o, Gq/11 and G12/13) for its downstream signalling. This study investigated the G protein subtypes responsible for the pro-inflammatory effect of TSH on macrophages. qPCR showed that Gi2, Gi3, Gas, Gq, G11, G12, G13, G15 are abundantly expressed by macrophages. The contribution of different G-protein pathways to the pro-inflammatory effect was studied by the corresponding inhibitors or siRNA interference. While TSH-induced IκB phosphorylation was not inhibited by Gs inhibitor NF449, Gi inhibitor pertussis toxin, Gq or G11 siRNA, it was blocked by phospholipase C inhibitor U73122 or G15 siRNA interference. TSH-induced ERK and P38 phosphorylation was blocked by G13 but not G12 siRNA interference. Interfering either G13 or G15 was able to block the pro-inflammatory effect of TSH on macrophages. The present study demonstrate that TSH activates macrophage inflammation by G13/ERK-P38/Rho GTPases and G15/PLC/PKCs/IκB pathways.

Activation of autophagy ameliorates age-related neurogenesis decline and neurodysfunction in adult mice

Adult neurogenesis is the ongoing generation of functional new neurons from neural progenitor cells (NPCs) in the mammalian brain. However, this process declines with aging, which is implicated in the recession of brain function and neurodegeneration. Understanding the mechanism of adult neurogenesis and stimulating it will benefit the mitigation of neurodegenerative diseases. Autophagy, a highly conserved process of cellular degradation, is essential for maintaining cellular homeostasis and normal function. Whether and how autophagy affects adult neurogenesis remains poorly understood. In present study, we revealed a close connection between impaired autophagy and adult neurogenetic decline. Expression of autophagy-related genes and autophagic activity were significantly declined in the middle-aged subventricular/subgranular zone (SVZ/SGZ) homogenates and cultured NPCs, and inhibiting autophagy by siRNA interference resulted in impaired pluripotency of NPCs. Conversely, stimulating autophagy by rapamycin not only revitalized the viability and pluripotency of middle-aged NPCs, but also facilitated the neurogenesis in middle-aged SVZ/SGZ. More importantly, autophagic activation by rapamycin also ameliorated the olfactory sensitivity and cognitional capacities in middle-aged mice. Taken together, our results reveal that compromised autophagy is involved in the decline of adult neurogenesis, which could be reversed by autophagy activation. It also shed light on the regulation of adult neurogenesis and paves the way for developing therapeutic strategy for aging and neurodegenerative diseases.

Expression of CDK9 in endometrial cancer tissues and its effect on the proliferation of HEC-1B

Endometrial cancer (EC) is one of the most common malignant tumors in the female reproductive system, which has been threatening the life and health of many women. Its incidence and mortality rate remain high, resulting in a low survival rate. In this study, the expression of cyclin-dependent kinase 9 (CDK9) in EC tissues was investigated, and its effect on the proliferation of EC cell line HEC-1B was preliminarily analyzed. RT-qPCR and Western blotting showed that CDK9 mRNA and protein were significantly reduced in the small interfering (si)RNA interference group compared with the siRNA control and blank control groups. MTT assay showed that the EC cell proliferative ability was significantly decreased, and phosphorylated-phosphatidylinositol 3 kinase (p-PI3K)/PI3K and phosphorylated-protein kinase B (p-AKT)/AKT were significantly reduced in the siRNA interference group compared with the siRNA control and blank control groups. In conclusion, the high expression of CDK9 is a factor of poor prognosis in EC, and the reduction of CDK9 can inhibit HEC-1B cell proliferation, which may be related to the inhibition on the activation of the PI3K/AKT signaling pathway.

Effect of siRNA Interference with Upregulated Gene 11 Expression on the Biological Functions of Osteosarcoma Cell Line MG63 and Their Mechanisms

This study investigated the effect of siRNA interference with URG11 expression on biological function of osteosarcoma cell line MG63 and its mechanism. MG63 cells cultured in vitro were apportioned to make up a control group (not transfected), NC-siRNA group (transfected nonspecific NC-siRNA), and URG11-siRNA group (transfected URG11-siRNA). The expression of URG11 was detected through reverse-transcription polymerase chain reaction (RT-PCR). The metastasis, infiltration, and apoptosis of MG63 cells were examined through CCK-8, Transwell, and flow cytometry techniques, respectively. The expressions of URG11 protein in each group of cells and of proteins β-catenin, c-Myc, cyclin D1, MMP-2, and survivin, which are related to the Wnt/β-catenin signal pathway, were visualized by the Western blot method. In comparison with the control group, transfected URG11-siRNA may reduce the expression levels of URG11 and URG11 mRNA, β-catenin, CyclinD1, c-Myc, MMP-2, and survivin proteins in MG63 cells, and may decrease the metastasis and infiltration of MG63 cells and enhance apoptosis; nevertheless, there was no obvious change in MG63 cells after NC-siRNA transfection. The proliferation and infiltration of MG63 cells can be inhibited by interference with URG11 expression, and the apoptosis of MG63 cells can be promoted by interference with URG11 expression. The mechanism of interference with URG11 expression may relate to inhibiting the activation of the Wnt/β-catenin pathway.

Androgen Receptor Regulates the Proliferation of Stretched Myoblasts and Its Mechanisms: IGF-1R-mediated p38 and ERK1/2 Pathways

BackgroundOur previous study has indicated that in C2C12 myoblasts androgen receptor (AR) plays a crucial role in 15% stretch-induced proliferation through insulin-like growth factor (IGF-1)-modulated PI3K/Akt, p38 and ERK1/2 pathways. In this study we further explored AR's effects on 15% and 20% stretches-modulated myoblasts proliferation and involved pathways in L6 (no detectable AR) and C2C12 (abundant AR) myoblasts.MethodsThe proliferation and the above indicators were compared between stretched L6 and C2C12 myoblasts by CCK8, ELISA and Western blot, then were detected again in stretched L6 myoblasts after transfection with AR overexpression plasmid utilizing LipoPlusTM 2000 reagent or treatment with different concentrations (200, 500, 1000 ng/ml) IGF-1 recombinant polypeptide.ResultsWe found that ①Under un-stretched states, the proliferation rate of L6 cells was slower than that of C2C12 cells, and the proliferation of C2C12 cells was repressed by AR knockdown using siRNA interference, while L6 myoblasts proliferation was promoted by overexpression AR. ②The proliferation rate as well as the activities of p38 and ERK1/2 increased by 15% stretch were much lower in L6 than that in C2C12 cells; AR overexpression enhanced the proliferation of 15% stretched L6 cells accompanied with increased activities of p38 and ERK1/2; ③20% stretch decreased the proliferation and activity of p38 in L6 more than that in C2C12 myoblasts; overexpression of AR totally reversed the anti-proliferation of 20% stretch and significantly enhanced p38 activity in L6 myoblasts;④In L6 myoblasts, overexpression AR increased IGF-1R expression despite no detectable IGF-1 was secreted; and recombinant IGF-1 increased not only the proliferation, but also the protein level of IGF-1R and activities of p38 and ERK1/2 in a dose-dependent manner in 15% stretched L6 myoblasts. ConclusionsAR fulfilled 15% stretch-induced pro-proliferation of myoblasts by activating IGF-1R-p38 and ERK1/2 pathways, while 20% stretch-induced anti-proliferation was mediated by inhibiting AR-IGF-1R-p38 pathway. This study is beneficial to understand in depth the role and mechanisms of AR on appropriate exercise increases while excessive exercise decreases muscle mass.

Oligodendrocyte Progenitor Cells Increase Chi3l1 Secretion in Exosome to Activate Myh9 and Promote Proliferation through Connexin47 Connected to Astrocytes

Background: Neurodegenerative diseases, caused by the loss of neurons or myelin sheath, are some of the most important neurological diseases that threaten the health of the elderly. In the CNS, oligodendrocytes (OLs) are the only cells that can form myelin. Astrocytes (ASTs) play a generally beneficial role in remyelination, including the proliferation and differentiation of oligodendrocyte precursor cells (OPCs) to OLs. However, the specific downstream mechanism is unclear.Methods: This study investigated the proliferation of OPCs in OPCs mono-culture, OPCs culture with ASTs supernatant, and ASTs-OPCs co-culture. Gene Ontology (GO) analysis were used to analyze the differentially expressed genes after transcriptome sequencing of these OPCs. Electron microscope, Nanoparticle Tracking Analysis (NTA), Fluorescence tracing of exosomes and Western blot were used to evaluate the effects of exsomes. Pull-down, co-immunoprecipitation (Co-IP) and mass spectrometry analys were conducted to find the downstream signal proliferation which is transmitted information into OPCs.Reasults: Direct contact co-culture of ASTs and OPCs promotes the proliferation of OPCs. After Cx47 siRNA interference under ASTs-OPCs co-culture, Chi3l1 secretion in exosome reveals associated decrease, and OPCs proliferation decreased. The cell proliferation induced by Chi3l1 was inhibited after siRNA interfered with Myh9, and the expression of cyclin D1 was also decreased.Conclusions: These results suggest that ASTs transmit information to OPCs by increasing gap junction channel Cx47, thereby promoting the secretion of Chi3l1 in exosome of OPCs. The secretory form of Chi3l1 in exosome might be easier to enter the target cell than in extracellular supernatant, which is beneficial to the activation of Myh9 to promote OPCs proliferation. This may be a potential target for drugs rescuing neurodegeneration diseases related to remyelination.

Oligodendrocyte Progenitor Cells Increase Chi3l1 Secretion in exosome to Activate Myh9 and Promote Proliferation through Connexin47 Connected to Astrocytes

Background: Neurodegenerative diseases, caused by the loss of neurons or myelin sheath, are some of the most important neurological diseases that threaten the health of the elderly. In the CNS, oligodendrocytes (OLs) are the only cells that can form myelin. Astrocytes (ASTs) play a generally beneficial role in remyelination, including the proliferation and differentiation of oligodendrocyte precursor cells (OPCs) to OLs. However, the specific downstream mechanism is unclear.Methods: This study investigated the proliferation of OPCs in OPCs mono-culture, OPCs culture with ASTs supernatant, and ASTs-OPCs co-culture. Gene Ontology (GO) analysis were used to analyze the differentially expressed genes after transcriptome sequencing of these OPCs. Electron microscope, Nanoparticle Tracking Analysis (NTA), Fluorescence tracing of exosomes and Western blot were used to evaluate the effects of exsomes. Pull-down, co-immunoprecipitation (Co-IP) and mass spectrometry analys were conducted to find the downstream signal proliferation which is transmitted information into OPCs.Reasults: Direct contact co-culture of ASTs and OPCs promotes the proliferation of OPCs. After Cx47 siRNA interference under ASTs-OPCs co-culture, Chi3l1 secretion in exosome reveals associated decrease, and OPCs proliferation decreased. The cell proliferation induced by Chi3l1 was inhibited after siRNA interfered with Myh9, and the expression of cyclin D1 was also decreased.Conclusions: These results suggest that ASTs transmit information to OPCs by increasing gap junction channel Cx47, thereby promoting the secretion of Chi3l1 in exosome of OPCs. The secretory form of Chi3l1 in exosome might be easier to enter the target cell than in extracellular supernatant, which is beneficial to the activation of Myh9 to promote OPCs proliferation. This may be a potential target for drugs rescuing neurodegeneration diseases related to remyelination.

LSH mediates gene repression through macroH2A deposition

The human Immunodeficiency Centromeric Instability Facial Anomalies (ICF) 4 syndrome is a severe disease with increased mortality caused by mutation in the LSH gene. Although LSH belongs to a family of chromatin remodeling proteins, it remains unknown how LSH mediates its function on chromatin in vivo. Here, we use chemical-induced proximity to rapidly recruit LSH to an engineered locus and find that LSH specifically induces macroH2A1.2 and macroH2A2 deposition in an ATP-dependent manner. Tethering of LSH induces transcriptional repression and silencing is dependent on macroH2A deposition. Loss of LSH decreases macroH2A enrichment at repeat sequences and results in transcriptional reactivation. Likewise, reduction of macroH2A by siRNA interference mimicks transcriptional reactivation. ChIP-seq analysis confirmed that LSH is a major regulator of genome-wide macroH2A distribution. Tethering of ICF4 mutations fails to induce macroH2A deposition and ICF4 patient cells display reduced macroH2A deposition and transcriptional reactivation supporting a pathogenic role for altered marcoH2A deposition. We propose that LSH is a major chromatin modulator of the histone variant macroH2A and that its ability to insert marcoH2A into chromatin and transcriptionally silence is disturbed in the ICF4 syndrome.

CircRNA-1926 Promotes the Differentiation of Goat SHF Stem Cells into Hair Follicle Lineage by miR-148a/b-3p/CDK19 Axis

Circular RNAs (CircRNAs) are a type of non-coding RNAs, which contain a covalently closed loop structure without 5′ to 3′ free ends. CircRNAs play essential roles in the regeneration of secondary hair follicle (SHF) and cashmere growth in goats. CircRNA-1926 was previously identified in SHF of cashmere goats, but its potential roles are unclear. In this study, we confirmed the expression of circRNA-1926 in SHF bulge of nine cashmere goats with a significantly higher level at anagen than that of telogen. Through the use of both overexpression and siRNA interference, we showed that circRNA-1926 promoted the differentiation of SHF stem cell into hair follicle lineage in cashmere goats which was evaluated via indictor genes Keratin 7 and Keratin 17. Using RNA pull-down, we found that circRNA-1926 bound with miR-148a/b-3p. Additionally, our data indicated that circRNA-1926 promoted the expression of the CDK19 gene. Using dual-luciferase reporter assays, it was revealed that circRNA-1926 positively regulated the CDK19 expression through miR-148a/b-3p. The results from this study demonstrated that circRNA-1926 contributes the differentiation of SHF stem cells into hair follicle lineages in cashmere goats via sponging miR-148a/b-3p to enhance CDK19 expression.