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Activin A, a member of the transforming growth factor-beta superfamily, plays a neuroprotective role in multiple neurological diseases. Endoplasmic reticulum (ER) stress-mediated apoptotic and autophagic cell death is implicated in a wide range of diseases, including cerebral ischemia and neurodegenerative diseases. Thapsigargin was used to induce PC12 cell death, and Activin A was used for intervention. Our results showed that Activin A significantly inhibited morphological changes in thapsigargin-induced apoptotic cells, and the expression of apoptosis-associated proteins [cleaved-caspase-12, C/EBP homologous protein (CHOP) and cleaved-caspase-3] and biomarkers of autophagy (Beclin-1 and light chain 3), and downregulated the expression of thapsigargin-induced ER stress-associated proteins [inositol requiring enzyme-1 (IRE1), tumor necrosis factor receptor-associated factor 2 (TRAF2), apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase (JNK) and p38]. The inhibition of thapsigargin-induced cell death was concentration-dependent. These findings suggest that administration of Activin A protects PC12 cells against ER stress-mediated apoptotic and autophagic cell death by inhibiting the activation of the IRE1-TRAF2-ASK1-JNK/p38 cascade.

Activin A (Act A), a member of transforming growth factor-β superfamily, plays a neuroprotective role in multiple neurological diseases through Act A/Smads signal activation. Traditionally, the up-regulation of Act A gene and extracellular Act A accumulation show the signal activation as a linear pathway. However, one of our discoveries indicated that Act A could lead a loop signaling in ischemic injury. To clarify the characteristic of this loop signaling in a non-pathological state, we up-regulated the expression of Act A, monitored extracellular Act A accumulation and examined the activity of Act A signaling, which was quantified by the expression of phosphorylated Smad3 and the fluorescence intensity of Smad4 in nuclei. The results demonstrated a noncanonical Act A signal loop with self-amplifying property in PC12 cells. Further, it showed self-limiting behavior due to temporary activation and spontaneous attenuation. This periodic behavior of Act A signal loop was found to be regulated by the level of Smad anchor for receptor activation (SARA). Moreover, increased activity of Act A signal loop could promote PC12 cell proliferation and enhance the survival rate of cells to Oxygen-Glucose Deprivation. These practical discoveries will bring new insight on the functional outcome of Act A signaling in neurological diseases by the further understanding: loop signaling.

Macrophages can be alternatively activated by TGF-β1 and high-ambient glucose, in which the role of Smad2 and the crosstalk between ERK and Smad2 pathways are not fully understood. The activation of ERK and Smad2 pathways and the expression of arginase-1 were detected by Western blot. The role of Smad2 and the relationship between ERK and Smad2 pathways were investigated by using biochemical inhibitors. The protein of arginase-1 was significantly overexpressed in RAW264.7 cells stimulated by TGF-β1 and high-ambient glucose, which can be partially blocked by not only U0126 (ERK inhibitor) but also SB431542 (Smad2 inhibitor). Furthermore, simply inhibiting one pathway had no effect on the other pathway. In conclusion, both ERK and Smad2 signal pathways are involved in the activation of macrophages induced by TGF-β1 and high-ambient glucose, while there is no crosstalk shown in the process.

Activin A (Act A), a member of the transforming growth factor-beta (TGF-β), reduces neuronal apoptosis during cerebral ischemia through Act A/Smads signaling pathway. However, little is known about the effect of Act A/Smads pathway on autophagy in neurons. Here, we found that oxygen-glucose deprivation (OGD)-induced autophagy was suppressed by exogenous Act A in a concentration-dependent manner and enhanced by Act A/Smads pathway inhibitor (ActRIIA-Ab) in neuronal PC12 cells. These results indicate that Act A/Smads pathway negatively regulates autophagy in OGD-treated PC12 cells. In addition, we found that c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways are involved in the OGD-induced autophagy. The activation of JNK and p38 MAPK pathways in OGD-treated PC12 cells was suppressed by exogenous Act A and enhanced by ActRIIA-Ab. Together, our results suggest that Act A/Smads signaling pathway negatively regulates OGD-induced autophagy via suppression of JNK and p38 MAPK pathways in neuronal PC12 cells.

Osteosarcoma is a type of malignant bone tumor with high metastasis and poor prognosis. Previous studies have demonstrated the involvement of LIM kinase 1 (LIMK1) in the proliferation of osteosarcoma cells. LIMK1 is overexpressed in human osteosarcoma tissues and cell lines. To further study LIMK1-associated mechanisms, we used shRNA targeted to the LIMK1 gene to block its expression in the osteosarcoma cell lines MG63 and U2OS. Insulin promoted the proliferation of MG63 cells in a time- and dose-dependent manner, however, this insulin induced proliferation was significantly inhibited by transfection of shRNA targeted to the LIMK1 gene, as well as by the PI3K inhibitor LY294002, but not by the mitogen‑activated protein kinase (MAPK) inhibitor PD98059. The level of cofilin phosphorylation was increased significantly following stimulation of insulin for 24 h, indicating the activation of LIMK1. MG63 cell proliferation was also significantly inhibited by 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) in a time-dependent manner. Furthermore, 1,25(OH)2D3 negated the inhibitory effect of LIMK1 shRNA, indicating that LIMK1 is important in the inhibitory pathway of 1,25(OH)2D3. The present study confirms that LIMK1 is important in regulating osteosarcoma cell proliferation via the insulin/PI3K/LIMK1 signaling pathway, thus the development of gene therapy for osteosarcoma targeting LIMK1 is warranted.

Adult cardiomyocytes (CMs) have very limited capacity to regenerate. Therefore, there is a great interest in developing strategies to treat infarcted CMs that are able to regenerate cardiac tissue and promote revascularization of infarcted zones in the heart. Recently, stem cell transplantation has been proposed to replace infarcted CMs and to restore the function of the affected tissue. This area of research has become very active in recent years due to the huge clinical need to improve the efficacy of currently available therapies. Slingshot (SSH) is a family of protein phosphatases, which can specifically dephosphorylate and reactivate cofilin and inhibit the polymerization of actin filaments and actively involved in cytoskeleton rearrangement. In this study, we found that SSH1L promoted morphology changes of microfilaments during differentiation but was inhibited by the inhibitors of actin polymerization such as cytochalasin D. Overexpression of SSH1L could promote cardiac-specific protein and genes expression. 5-Aza can induce the differentiation of hMSCs into cardiomyocyte-like cells in vitro. We also observed that SSH1L efficiently promotes hMSCs differentiation into cardiomyocyte-like cells through regulation and rearrangement of cytoskeleton. Our work provides evidence that supports the positive role of SSH1L in the mechanism of stem cell differentiation into cardiomyocyte-like cells.

In this study, the effect of aucubin on H(2)O(2)-induced apoptosis was studied by using a rat pheochromocytoma (PC12) cell line. We have analyzed the apoptosis of H(2)O(2)-induced PC12 cells, H(2)O(2)-induced apoptosis appeared to correlate with lower Bcl-2 expression, higher Bax expression and sequential activation of caspase-3 leading to cleavage of poly-ADP-ribose polymerase (PARP). Aucubin not only inhibited lower Bcl-2 expression, high Bax expression, but also modulated caspase-3 activation, PARP cleavage, and eventually protected against H(2)O(2)-induced apoptosis. These results indicated that aucubin can obstruct H(2)O(2)-induced apoptosis by regulating of the expression of Bcl-2 and Bax, as well as suppression of caspases cascade activation.

This meta-analysis was conducted to assess the association of Megsin 2093C/T, 2180C/T, C25663G gene polymorphism with the risk of IgA nephropathy (IgAN). The association literatures were identified from PubMed, Embase, Cochrane Library and CBM-disc (China Biological Medicine Database) on 1 January 2014, and eligible reports were recruited and synthesized. Seven eligible reports were recruited into this meta-analysis for the association of Megsin 2093C/T, 2180C/T, C25663G gene polymorphism with IgAN risk. In this meta-analysis, the association of Megsin 2093C/T TT genotype with IgAN risk in Asians was found. Interestingly, Megsin C25663G G allele and GG genotype were associated with the risk of IgAN in Asian population. However, Megsin 2180C/T gene polymorphism was not associated with IgAN risk in Asians. In conclusion, Megsin 2093C/T TT genotype, and C25663G G allele and GG genotype were associated with the risk of IgAN in Asian population. However, more studies should be performed in the future to confirm this association.

Mammalian skeletal muscle can remodel, repair, and regenerate itself by mobilizing satellite cells, a resident population of myogenic progenitor cells. Muscle injury and subsequent activation of myogenic progenitor cells is associated with oxidative stress. Cytoglobin is a hemoprotein expressed in response to oxidative stress in a variety of tissues, including striated muscle. In this study, we demonstrate that cytoglobin is up-regulated in activated myogenic progenitor cells, where it localizes to the nucleus and contributes to cell viability. siRNA-mediated depletion of cytoglobin from C2C12 myoblasts increased levels of reactive oxygen species and apoptotic cell death both at baseline and in response to stress stimuli. Conversely, overexpression of cytoglobin reduced reactive oxygen species levels, caspase activity, and cell death. Mice in which cytoglobin was knocked out specifically in skeletal muscle were generated to examine the role of cytoglobin in vivo. Myogenic progenitor cells isolated from these mice were severely deficient in their ability to form myotubes as compared with myogenic progenitor cells from wild-type littermates. Consistent with this finding, the capacity for muscle regeneration was severely impaired in mice deficient for skeletal-muscle cytoglobin. Collectively, these data demonstrate that cytoglobin serves an important role in muscle repair and regeneration.

Cofilin promotes actin filament turnover by severing and depolymerizing actin filaments. Cofilin is inactivated by phosphorylation on Ser-3 by LIM-kinase1 (LIMK1) and is activated when protein phosphatase Slingshot-1L (SSH1L) dephosphorylates this residue. The authors have shown that Ca 2+ -induced cofilin dephosphorylation is mediated by calcineurin (Cn)-dependent activation of SSH1L. In this study, Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is shown to negatively regulate SSH1L activity and bind to SSH1L in a complex with 14-3-3. Phosphorylation of LIMK1 by CaMKII and its subsequent activation regulates the subcellular localization of SSH1L. Based on these findings, the authors suggest that CaMKII and Cn provide a switch-like mechanism that controls Ca 2+ -dependent LIMK1, SSH1L and cofilin activation, and subsequently actin cytoskeletal reorganization.

SUMMARY Cytoglobin (Cygb) is a novel tissue hemoprotein relatively similar to myoglobin (Mb). Because Cygb shares several structural features with Mb, we hypothesized that Cygb functions in the modulation of oxygen and nitric oxide metabolism or in scavenging free radicals within a cell. In the present study we examined the spatial and temporal expression pattern of Cygb during murine embryogenesis. Using in situ hybridization, RT-PCR, and Northern blot analyses, limited Cygb expression was observed during embryogenesis compared with Mb expression. Cygb expression was primarily restricted to the central nervous system and neural crest derivatives during the latter stages of development. In the adult mouse, Cygb is expressed in distinct regions of the brain as compared with neuroglobin (Ngb), another globin protein, and these regions are responsive to oxidative stress (i.e., hippocampus, thalamus, and hypothalamus). In contrast to Ngb, Cygb expression in the brain is induced in response to chronic hypoxia (10% oxygen). These results support the hypothesis that Cygb is an oxygen-responsive tissue hemoglobin expressed in distinct regions of the normoxic and hypoxic brain and may play a key role in the response of the brain to a hypoxic insult. (J Histochem Cytochem 54:1349–1361, 2006)