Caspase‐3‐mediated cyclic stretch‐induced myoblast apoptosis via a Fas/FasL‐independent signaling pathway during myogenesis

Skeletal muscle cells are exposed to mechanical stretch during embryogenesis. Increased stretch may contribute to cell death, and the molecular regulation by stretch remains incompletely understood. The aim of this study was to investigate the effects of cyclic stretch on cell death and apoptosis in myoblast using a Flexercell Strain Unit. Apoptosis was studied by annexin V binding and PI staining, DNA size analysis, electron microphotograph, and caspase assays. Fas/FasL expression was determined by Western blot. When myoblasts were cultured on a flexible membrane and subjected to cyclic strain stress, apoptosis was observed in a time‐dependent manner. We also determined that stretch induced cleavage of caspase‐3 and increased caspase‐3 activity. Caspase‐3 inhibition reduced stretch‐induced apoptosis. Protein levels of Fas and FasL remained unchanged. Our findings implicated that stretch‐induced cell death is an apoptotic event, and that the activation of caspase cascades is required in stretch‐induced cell apoptosis. Furthermore, we had provided evidence that caspase‐3 mediated cyclic stretch‐induced myoblast apoptosis. Mechanical forces induced activation of caspase‐3 via signaling pathways independent of Fas/FasL system. J. Cell. Biochem. 107: 834–844, 2009. © 2009 Wiley‐Liss, Inc.     

Insulin‐like growth factor‐I prevents caspase‐mediated apoptosis in Schwann cells

Both neurons and glia succumb to programmed cell death (PCD) when deprived of growth factors at critical periods in development or following injury. Insulin‐like growth factor‐I (IGF‐I) prevents apoptosis in neurons in vitro. To investigate whether IGF‐I can protect Schwann cells (SC) from apoptosis, SC were harvested from postnatal day 3 rats and maintained in serum‐containing media until confluency. When cells were switched to serum‐free defined media (DM) for 12–72 h, they underwent PCD. Addition of insulin or IGF‐I prevented apoptosis. Bisbenzamide staining revealed nuclear condensation and formation of apoptotic bodies in SC grown in DM alone, but SC grown in DM plus IGF‐I had normal nuclear morphology. The phosphatidylinositol 3‐kinase (PI 3‐K) inhibitor LY294002 blocked IGF‐I–mediated protection. Caspase‐3 activity was rapidly activated upon serum withdrawal in SC, and the caspase inhibitor BAF blocked apoptosis. These results suggest that IGF‐I rescues SC from apoptosis via PI 3‐K signaling which is upstream from caspase activation. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 540–548, 1999     

Molecular features of neural stem cells enable their enrichment using pharmacological inhibitors of survival‐promoting kinases

Isolating a pure population of neural stem cells (NSCs) has been difficult since no exclusive surface markers have been identified for panning or FACS purification. Moreover, additional refinements for maintaining NSCs in culture are required, since NSCs generate a variety of neural precursors (NPs) as they proliferate. Here, we demonstrate that post‐natal rat NPs express low levels of pro‐apoptotic molecules and resist phosphatidylinositol 3′OH kinase and extracellular regulated kinase 1/2 inhibition as compared to late oligodendrocyte progenitors. Furthermore, maintaining subventricular zone precursors in LY294002 and PD98059, inhibitors of PI3K and ERK1/2 signaling, eliminated lineage‐restricted precursors as revealed by enrichment for Nestin⁺/SOX‐2⁺ cells. The cells that survived formed neurospheres and 89% of these neurospheres were tripotential, generating neurons, astrocytes, and oligodendrocytes. Without this enrichment step, less than 50% of the NPs were Nestin⁺/SOX‐2⁺ and 42% of the neurospheres were tripotential. In addition, neurospheres enriched using this procedure produced 3‐times more secondary neurospheres, supporting the conclusion that this procedure enriches for NSCs. A number of genes that enhance survival were more highly expressed in neurospheres compared to late oligodendrocyte progenitors. Altogether, these studies demonstrate that primitive neural precursors can be enriched using a relatively simple and inexpensive means that will facilitate cell replacement strategies using stem cells as well as other studies whose goal is to reveal the fundamental properties of primitive neural precursors.

     

Caspase‐8: a key role in the pathogenesis of diabetic embryopathy

Maternal diabetes causes neural tube defects in embryos, which are associated with increased apoptosis in the neuroepithelium. Many factors, including effector caspases, have been shown to be involved in the events. However, the key regulators have not been identified and the underlying mechanisms remain to be addressed. Caspase‐8, an initiator caspase, has been shown to be altered in diabetic embryopathy, suggesting a role as an upstream apoptotic regulator. Using mouse embryos as a model system, this study demonstrates that caspase‐8 is required for the production of hyperglycemia‐associated embryonic malformations. Caspase‐8 was shown to be expressed in the developing neural tube. Its activity, as evidenced by enhanced cleavage, was increased by hyperglycemia. These changes were associated with increased formation of the active cleavage of Bid. Inhibition of caspase‐8 activity in high glucose–challenged embryos reduced the rate of embryonic malformation and this was associated with decreased apoptosis in the neuroepithelium of the neural tube. Inhibition of caspase‐8 activity also reduced hyperglycemia‐induced Bid activation and caspase‐9 cleavage. These data suggest that caspase‐8 may control diabetic embryopathy‐associated apoptosis via regulation of the Bid‐stimulated mitochondrion/caspase‐9 pathway. Birth Defects Res (Part B)86:72‐77, 2009. ©2009 Wiley‐Liss, Inc.     

Senescence and odontoblastic differentiation of dental pulp cells

Cellular senescence has been suggested to be involved in physiological changes of cytokine production. Previous studies showed that the concentration of tumor necrosis factor-α (TNF-α) is higher in the blood of aged people compared with that of young people. So far, the precise effects of TNF-α on the odontoblastic differentiation of pulp cells have been controversial. Therefore, we aimed to clarify how this cytokine affected pulp cells during aging. Human dental pulp cells (HDPCs) were cultured until reaching the plateau of their growth, and the cells were isolated at actively (young HDPCs; yHDPCs) or inactively (senescent HDPCs; sHDPCs) proliferating stages. sHDPCs expressed senescence-related molecules while yHDPCs did not. When these HDPCs were cultured in an odontoblast-inductive medium, both young and senescent cells showed mineralization, but mineralization in sHDPCs was lower compared with yHDPCs. However, the administration of TNF-α to this culture medium altered these responses: yHDPCs showed downregulated mineralization, while sHDPCs exhibited significantly increased mineralization. Furthermore, the expression of tumor necrosis factor receptor 1 (TNFR1), a receptor of TNF-α, was significantly upregulated in sHDPCs compared with yHDPCs. Downregulation of TNFR1 expression led to decreased mineralization of TNF-α-treated sHDPCs, whereas restored the reduction in TNF-α-treated yHDPCs. These results suggested that sHDPCs preserved the odontoblastic differentiation capacity and TNF-α promoted odontoblastic differentiation of HDPCs with the progress of their population doublings through increased expression of TNFR1. Thus, TNF-α might exert a different effect on the odontoblastic differentiation of HDPCs depending on their proliferating activity. In addition, the calcification of pulp chamber with age may be related with increased reactivity of pulp cells to TNF-α.     

Matrigel-induced acinar differentiation is followed by apoptosis in HSG cells

It has been shown that a human salivary gland cell line (HSG) is capable of differentiation into gland-like structures, though little is known of how morphological features are formed or controlled. Here we investigated the changes in cell proliferation and apoptosis upon terminal differentiation of HSG cells in Matrigel, an extracellular matrix derivative. Changes in the expression of survivin, a prominent anti-apoptotic factor, and caspase-3, a key apoptotic factor were also measured. In order to better understand the involvement of key signal transduction pathways in this system we pharmacologically blocked the activity of tyrosine kinase, nuclear factor kappa B(NF kappa B), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K) and matrix metalloproteases (MMP). Results of these studies demonstrate that cytodifferentiation of HSG cells to an acinar phenotype is accompanied first by a decrease of cell proliferation and then by a massive programmed cell death, affected by multiple signal transduction pathways. Thus, Matrigel alone is insufficient for the full maturation and long term survival of the newly formed acini: the presence of other factors is necessary to complete the acinar differentiation of HSG cells.     

High susceptibility of neural stem cells to methylmercury toxicity: effects on cell survival and neuronal differentiation

Neural stem cells (NSCs) play an essential role in both the developing embryonic nervous system through to adulthood where the capacity for self-renewal may be important for normal function of the CNS, such as in learning, memory and response to injury. There has been much excitement about the possibility of transplantation of NSCs to replace damaged or lost neurones, or by recruitment of endogenous precursors. However, before the full potential of NSCs can be realized, it is essential to understand the physiological pathways that control their proliferation and differentiation, as well as the influence of extrinsic factors on these processes. In the present study we used the NSC line C17.2 and primary embryonic cortical NSCs (cNSCs) to investigate the effects of the environmental contaminant methylmercury (MeHg) on survival and differentiation of NSCs. The results show that NSCs, in particular cNSCs, are highly sensitive to MeHg. MeHg induced apoptosis in both models via Bax activation, cytochrome c translocation, and caspase and calpain activation. Remarkably, exposure to MeHg at concentrations comparable to the current developmental exposure (via cord blood) of the general population in many countries inhibited spontaneous neuronal differentiation of NSCs. Our studies also identified the intracellular pathway leading to MeHg-induced apoptosis, and indicate that NSCs are more sensitive than differentiated neurones or glia to MeHg-induced cytotoxicity. The observed effects of MeHg on NSC differentiation offer new perspectives for evaluating the biological significance of MeHg exposure at low levels.     

Caspase‐1 inflammasome activity in patients with Staphylococcus aureus bacteremia

The inflammasome is a multiprotein complex that mediates caspase‐1 activation with subsequent maturation of the proinflammatory cytokines IL‐1β and IL‐18. The NLRP3 inflammasome is known to be activated by Staphylococcus aureus, one of the leading causes of bacteremia worldwide. Inflammasome activation and regulation in response to bacterial infection have been found to be of importance for a balanced host immune response. However, inflammasome signaling in vivo in humans initiated by S. aureus is currently sparsely studied. This study therefore aimed to investigate NLRP3 inflammasome activity in 20 patients with S. aureus bacteremia (SAB), by repeated measurement during the first week of bacteremia, compared with controls. Caspase‐1 activity was measured in monocytes and neutrophils by flow cytometry detecting FLICA (fluorescent‐labeled inhibitor of caspase‐1), while IL‐1β and IL‐18 was measured by Luminex and ELISA, respectively. As a measure of inflammasome priming, messenger RNA (mRNA) expression of NLRP3, CASP1 (procaspase‐1), and IL1B (pro‐IL‐1β) was analyzed by quantitative PCR. We found induced caspase‐1 activity in innate immune cells with subsequent release of IL‐18 in patients during the acute phase of bacteremia, indicating activation of the inflammasome. There was substantial interindividual variation in caspase‐1 activity between patients with SAB. We also found an altered inflammasome priming with low mRNA levels of NLRP3 accompanied by elevated mRNA levels of IL1B. This increased knowledge of the individual host immune response in SAB could provide support in the effort to optimize management and treatment of each individual patient.     

STIP overexpression confers oncogenic potential to human non‐small cell lung cancer cells by regulating cell cycle and apoptosis

Sip1/tuftelin‐interacting protein (STIP), a multidomain nuclear protein, is a novel factor associated with the spliceosome, yet its role and molecular function in cancer remain unknown. In this study, we show, for the first time, that STIP is overexpressed in non‐small cell lung cancer (NSCLC) tissues compared to adjacent normal lung tissues. The depletion of endogenous STIP inhibited NSCLC cell proliferation in vitro and in vivo, caused cell cycle arrest and induced apoptosis. Cell cycle arrest at the G2/M phase was associated with the expression and activity of the cyclin B1‐CDK1 (cyclin‐dependent kinase 1) complex. We also provide evidence that STIP knockdown induced apoptosis by activating both caspase‐9 and caspase‐3 and by altering the Bcl‐2/Bax expression ratio. RNA sequencing data indicated that the MAPK mitogen‐activated protein kinases, Wnt, PI3K/AKT, and NF‐κB (nuclear factor kappa‐light‐chain‐enhancer of activated B cells) signalling pathways might be involved in STIP‐mediated tumour regulation. Collectively, these results suggest that STIP may be a novel potential diagnostic and therapeutic target for NSCLC.     

Follicle‐stimulating hormone impairs dental pulp stem cells odontogenic differentiation

In addition to bone, the dentin‐pulp complex is also influenced by menopause, showing a decreased regenerative capacity. High levels of follicle‐stimulating hormone (FSH) during menopause could directly regulate bone metabolism. Here, the role of FSH in the odontogenic differentiation of the dentin‐pulp complex was investigated. Dental pulp stem cells (DPSCs) were isolated. CCK‐8 assays, cell apoptosis assays, Western blotting (WB), real‐time RT‐PCR, alkaline phosphatase activity assays, and Alizarin Red S staining were used to clarify the effects of FSH on the proliferation, apoptosis and odontogenic differentiation of the DPSCs. MAPK pathway‐related factors were explored by WB assays. FSH and its inhibitor were used in OVX rats combined with a direct pulp‐capping model. HE and immunohistochemistry were used to detect reparative dentin formation and related features. The results indicated that FSH significantly decreased the odontogenic differentiation of the DPSCs without affecting cell proliferation and apoptosis. Moreover, FSH significantly activated the JNK signalling pathway, and JNK inhibitor partly rescued the inhibitory effect of FSH on DPSC differentiation. In vivo, FSH treatment attenuated the dentin bridge formation and mineralization‐related protein expression in the OVX rats. Our findings indicated that FSH reduced the odontogenic capacity of the DPSCs and was involved in reparative dentinogenesis during menopause.     

Epigallocatechin‐3‐gallate suppresses the lipid deposition through the apoptosis during differentiation in bovine bone marrow mesenchymal stem cells

Epigallocatechin gallate (EGCG), a major component of tea, has known effects on obesity, fatty liver, and obesity‐related cancer. We explored the effects of EGCG on the differentiation of bovine mesenchymal stem cells (BMSCs, which are multipotent) in a dose‐ and time‐dependent manner. Differentiating BMSCs were exposed to various concentrations of EGCG (0, 10, 50, 100, and 200 µM) for 2, 4, and 6 days. BMSCs were cultured in Dulbecco's modified Eagle's medium (DMEM)/high‐glucose medium with adipogenic inducers for 6 days, and the expression levels of various genes involved in adipogenesis were measured using real‐time polymerase chain reaction (PCR) and Western blotting. We assessed apoptosis by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) staining of control and EGCG‐exposed cells. We found that EGCG significantly suppressed fat deposition and cell viability (P < 0.05). The mRNA and protein levels of various adipogenic factors were measured. Expression of the genes encoding peroxisome proliferator‐activated receptor gamma (PPARG), CCAAT/enhancer‐binding protein alpha (CEBPA), fatty acid‐binding protein 4 (FABP4), and stearoyl‐CoA desaturase (SCD) were diminished by EGCG during adipogenic differentiation (P < 0.05). We also found that EGCG lowered the expression levels of the adipogenic proteins encoded by these genes (P < 0.05). EGCG induced apoptosis during adipogenic differentiation (P < 0.05). Thus, exposure to EGCG potentially inhibits adipogenesis by triggering apoptosis; the data suggest that EGCG inhibits adipogenic differentiation in BMSCs.     

Effect of glypican‐1 gene on the pulp cells during the reparative dentine process

GPC‐1 (glypican‐1) is a cell surface heparan sulfate proteoglycan that acts as a co‐receptor for heparin‐binding growth factors and members of the TGF‐β (transforming growth factor beta‐1) family. The function of cell‐surface proteoglycans in the reparative dentine process has been under investigation. Gpc‐1 was detected with similar frequency as tgf‐β1 in the cDNA library using mRNA from the odontoblast‐like cell‐enriched pulp of rat incisors. The aim of this study was to test our hypothesis that gpc‐1 may be related to reparative dentine formation. We examined the expression of this gene during the reparative dentine process, as well as the effect of gpc‐1 on odontoblast‐like cell differentiation using siRNA (small interfering RNA) to down‐regulate gpc‐1 expression. Immunohistological examination showed that GPC‐1 was expressed in pulp cells entrapped by fibrodentine and odontoblast‐like cells as well as TGF‐β1. The mRNAs for gpc‐1, ‐3 and ‐4, except for gpc‐2, were expressed during odontoblast‐like cell differentiation in pulp cells. The relative levels of gpc‐1 mRNA were increased prior to the differentiation stages and were decreased during the secretory and maturation stages of pulp cells. Down‐regulation of gpc‐1 expression resulted in a 3.9‐fold increase in tgf‐β1 expression in pulp cells and a 0.3‐fold decrease in dspp (dentine sialophosphoprotein) expression compared with control. These results suggested that gpc‐1 and tgfβ‐1 expression are necessary for the onset of differentiation, but should be down‐regulated before other molecules are implicated in the formation of reparative dentine. In conclusion, gpc‐1 expression in odontoblast‐like cells is associated with the early differentiation but not with the formation of reparative dentine.