Sheau-Huei Chueh

Sphingosylphosphorylcholine stimulates mitogen-activated protein kinase via a Ca2+-dependent pathway

In cultured porcine aortic smooth muscle cells, sphingosylphosphorylcholine (SPC), ATP, or bradykinin (BK) induced a rapid dose-dependent increase in the cytosolic Ca2+ concentration ([Ca2+]i) and also stimulated inositol 1,4,5-trisphosphate (IP3) generation. Pretreatment of cells with pertussis toxin blocked the SPC-induced IP3 generation and [Ca2+]iincrease but had no effect on the action of ATP or BK. In addition, SPC stimulated the mitogen-activated protein kinase (MAPK) and increased DNA synthesis, whereas neither ATP nor BK produced such effects. Both the SPC-induced MAPK activation and DNA synthesis were pertussis toxin sensitive. SPC-induced MAPK activation was blocked by treatment of cells with the phospholipase C inhibitor, U-73122, or the intracellular Ca2+-ATPase inhibitor, thapsigargin, but not by removal of extracellular Ca2+. Lysophosphatidic acid induced cellular responses similar to SPC in a pertussis toxin-sensitive manner in terms of [Ca2+]iincrease, IP3 generation, MAPK activation, and DNA synthesis. Platelet-derived growth factor (PDGF) also induced a [Ca2+]iincrease, MAPK activation, and DNA synthesis in the same cells; however, the PDGF-induced MAPK activation was not sensitive to pertussis toxin and changes in [Ca2+]i. SPC-induced MAPK activation was inhibited by pretreatment of cells with staurosporine, W-7, or calmidazolium. Our results suggest that, in porcine aortic smooth muscle cells, MAPK is not activated by the increase in [Ca2+]iunless a pertussis toxin-sensitive G protein is simultaneously stimulated, indicating the role of Ca2+ in pertussis toxin-sensitive G protein-mediated MAPK activation.

Calcium mobilization from the intracellular mitochondrial and nonmitochondrial stores of the rat cerebellum

Two major intracellular Ca2+ stores, the mitochondrial and nonmitochondrial (microsomes) fractions isolated from rat cerebellum exhibited a Ca2+ concentration and ATP-dependent Ca2+ accumulation. The maximal Ca2+ accumulation in mitochondria was higher than in microsomes, but the affinity of the mitochondria for Ca2+ was lower. In this study, Ca2+ accumulation within the mitochondria was energized by ATP hydrolysis. Thus, the protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and the F1F0 ATP synthase inhibitor, oligomycin, blocked Ca2+ accumulation and induced the discharge of the entrapped Ca2+ in the mitochondria, whereas the metabolic inhibitor, rotenone, affected neither the Ca2+ accumulation nor discharge. On the other hand, the uniporter inhibitor, ruthenium red, blocked the mitochondrial accumulation of Ca2+, but did not cause the discharge of preloaded Ca2+. In addition, arachidonic acid (AA), sphingosylphosphorylcholine (SPC) and sphingosine (SPH) elicited the dose-dependent release of Ca2+ from microsomal stores. Although the magnitudes of the Ca2+ release induced by AA, SPC or SPH were all dependent on the presence of extravesicular Ca2+ at concentrations ranging from 0.01 to 0.1 microM Ca2+, only the AA- and SPC-evoked Ca2+ releases were insensitive to temperature. The mitochondria were more sensitive than the microsomes to the AA induced release of accumulated Ca2+. Our results indicate the existence of multiple intracellular Ca2+ stores in nerve cells which can be released by various Ca2+ mediators.

Effects of interleukin-15 on neuronal differentiation of neural stem cells.

Interleukin-15 (IL-15) signaling has pleiotropic actions in many cell types during development and has been best studied in cells of immune system lineage, where IL-15 stimulates proliferation of cytotoxic T cells and induces maturation of natural killer cells. A few reports have indicated that IL-15 and the IL-15 receptor are expressed in central nervous system tissues and neuronal cell lines. Because this aspect of IL-15 action is poorly studied, we used cultured rat neural stem cells (NSCs) to study IL-15 signal transduction and activity. Primary cultures of rat NSCs in culture will form neurospheres and will differentiate into neuron, astrocyte, and oligodendrocyte progenitors under permissive conditions. We found by immunofluorescence that the IL-15Ralpha subunit of the IL-15 receptor was expressed in NSCs and differentiating neurons, but not astrocyte or oligodendrocyte progenitors. We also showed that IL-15 treatment reduced MAP-2 protein levels in neurons and could reduce neurite outgrowth in differentiating neurons but did not affect NSC proliferation, and cell proportions and viability of the corresponding lineage cells. In the presence of a STAT3 inhibitor, Stattic, IL-15 no longer reduced MAP-2 protein levels. IL-15 treatment caused STAT3 phosphorylation. Furthermore, using anti-IL-15Ralpha antibody to block IL-15 signaling completely inhibited IL-15-induced phosphorylation of STAT3 and prevented IL-15 from decreasing neurite outgrowth. In conclusion, IL-15 may influence neural cell differentiation through a signal transduction pathway involving IL-15Ralpha and STAT3. This signal transduction modifies MAP-2 protein levels and, consequently, the differentiation of neurons from NSCs, as evidenced by reduced neurite outgrowth.

Involvement of SHP2 in focal adhesion, migration and differentiation of neural stem cells

OBJECTIVES SHP2 (Src-homology-2 domain-containing protein tyrosine phosphatase) plays an important role in cell adhesion, migration and cell signaling. However, its role in focal adhesion, differentiation and migration of neural stem cells is still unclear. METHODS In this study, rat neurospheres were cultured in suspension and differentiated neural stem cells were cultured on collagen-coated surfaces. RESULTS The results showed that p-SHP2 co-localized with focal adhesion kinase (FAK) and paxillin in neurospheres and in differentiated neural precursor cells, astrocytes, neurons, and oligodendrocytes. Suppression of SHP2 activity by PTP4 or siRNA-mediated SHP2 silencing caused reduction in the cell migration and neurite outgrowth, and thinning of glial cell processes. Differentiation-induced activation of FAK, Src, paxillin, ERK1/2, and RhoA was decreased by SHP2 inactivation. CONCLUSIONS These results indicate that SHP2 is recruited in focal adhesions of neural stem cells and regulates focal adhesion formation. SHP2-mediated regulation of neural differentiation and migration may be related to formation of focal adhesions and RhoA and ERK1/2 activation.

Inhibition of the mammalian target of rapamycin promotes cyclic AMP-induced differentiation of NG108-15 cells

To clarify the involvement of autophagy in neuronal differentiation, the effect of rapamycin, an mTOR complex inhibitor, on the dibutyryl cAMP (dbcAMP)-induced differentiation of NG108-15 cells was examined. Treatment of NG108-15 cells with 1 mM dbcAMP resulted in induction of differentiation, including neurite outgrowth and varicosity formation, enhanced voltage-sensitive Ca2+ channel activity and expression of microtubule-associated protein 2, and these effects involved phosphorylation of cAMP-response element binding protein (CREB) and extracellular signal regulated kinase (ERK). Simultaneous application of dbcAMP and rapamycin synergistically increased and accelerated differentiation. mTOR or raptor silencing with siRNA had a similar effect to rapamycin. Rapamycin and silencing of mTOR or raptor evoked autophagy, while blockade of autophagy by addition of 3-methyladenine or beclin 1 or Atg5 silencing prevented the potentiation of differentiation. Silencing of rictor also evokes autophagy, at a level 55% of that induced by raptor silencing and enhancement of differentiation is proportional. Rapamycin also caused increased ATP generation and cell cycle arrest in G0/G1 phase, but had no effect on CREB and ERK phosphorylation. dbcAMP also induced ATP generation, but not autophagy or cell cycle arrest. These results suggest that the increased autophagy, ATP generation and cell cycle arrest caused by mTOR inhibition promotes the dbcAMP-induced differentiation of NG108-15 cells.

Dual effect of capsaicin on cell death in human osteosarcoma G292 cells.

Thirty percent of osteosarcoma patients die within 5 years. New agents that induce apoptosis of osteosarcoma cells might be therapeutically useful. Here, we characterized the apoptotic mechanism induced by capsaicin in G292 osteosarcoma cells. Our results show that capsaicin induces an increase in the cytosolic Ca(2+) concentration which is independent of the extracellular Ca(2+) concentration and depletes intracellular Ca(2+) stores, suggesting the presence of endoplasmic reticulum transient receptor potential vanilloid receptor type 1. Capsaicin also activates the mitochondrial caspase 3-dependent death cascade. Rapamycin, an inhibitor of mammalian target of rapamycin, evokes autophagy, as do capsaicin or thapsigargin, a sarco(endo)plasmic reticulum Ca(2+) ATPase inhibitor that causes Ca(2+) store depletion. Capsaicin-induced cell death is completely inhibited by co-treatment with the pan-caspase inhibitor Z-VAD-fmk and increased by the autophagy inhibitor 3-methyladenine, suggesting the existence of an autophagy-dependent anti-apoptotic mechanism. Capsaicin also induces ERK phosphorylation, which acts as a downstream effector of autophagy. 3-Methyladenine or PD98059, an ERK kinase inhibitor, restores capsaicin-induced cell death in the presence of Z-VAD-fmk, suggesting that inhibition of autophagy activates a second cell death pathway that is caspase-independent. Taken together, our data show that capsaicin causes Ca(2+) depletion of intracellular Ca(2+) stores and simultaneously activates the mitochondrial caspase-dependent death cascade and autophagy-dependent ERK activation and that the latter counteracts a second death signaling pathway that is caspase-independent.

Baicalein Decreases Hydrogen Peroxide-Induced Damage to NG108-15 Cells via Upregulation of Nrf2.

Baicalein is a flavonoid inhibitor of 12‐lipoxygenase. Here, we investigated its effect on hydrogen peroxide‐induced damage to NG108–15 cells. Hydrogen peroxide activated the mitochondrial apoptotic pathway, decreased Nrf2 expression, increased reactive oxygen species (ROS) levels, reduced viability, and increased cell death after 2–24 h treatment of NG108–15 cells. Co‐treatment with hydrogen peroxide and baicalein completely suppressed the activation of mitochondrial apoptotic pathway by upregulating Nrf2 expression and reducing ROS stress and partially inhibited the effects on cell viability and cell death. Silencing of 12‐lipoxygenase had a similar protective effect to baicalein on hydrogen peroxide‐induced damage by blocking the hydrogen peroxide‐induced decrease in Nrf2 expression and increase in ROS levels. Neither protective effect was altered by addition of 12‐hydroxyeicosatetraenoic acid, the product of 12‐lipoxygenase, suggesting that hydrogen peroxide induced damage via 12‐lipoxygenase by another, as yet unknown, mechanism, rather than activating it. Co‐treatment of cells with hydrogen peroxide and N‐acetylcysteine or the Nrf2 inducer sulforaphane reduced hydrogen peroxide‐induced damage in a similar fashion to baicalein, while the Nrf2 inhibitor retinoic acid blocked the protective effect of baicalein. Silencing Nrf2 also inhibited the protective effects of baicalein, sulforaphane, and N‐acetylcysteine and resulted in high ROS levels, suggesting ROS elimination was mediated by Nrf2. Taken together our results suggest that baicalein protects cells from hydrogen peroxide‐induced activation of the mitochondrial apoptotic pathway by upregulating Nrf2 and inhibiting 12‐lipoxygenase to block the increase in ROS levels. Hydrogen peroxide also activates a second mitochondrial dysfunction independent death pathway which is resistant to baicalein. J. Cell. Physiol. 230: 1840–1851, 2015.

Mesenchymal stem cells from rat olfactory bulbs can differentiate into cells with cardiomyocyte characteristics.

Mesenchymal stromal/stem cells (MSCs) are widely distributed in different tissues such as bone marrow, adipose tissues, peripheral blood, umbilical cord and amnionic fluid. Recently, MSC‐like cells were also found to exist in rat olfactory bulb and are capable of inducing differentiation into mesenchymal lineages – osteocytes, chondrocytes and adipocytes. However, whether these cells can differentiate into myocardial cells is not known. In this study, we examined whether olfactory bulb‐derived MSCs could differentiate into myocardial cells in vitro. Fibroblast‐like cells isolated from the olfactory bulb of neonatal rats were grown under four conditions: no treatment; in the presence of growth factors (neuregulin‐1, bFGF and forskolin); co‐cultured with cardiomyocytes; and co‐cultured with cardiomyocytes plus neuregulin‐1, bFGF and forskolin. Cell differentiation into myocardial cells was monitored by RT–PCR, light microscopy immunofluorescence, western blot analysis and contractile response to pharmacological treatments. The isolated olfactory bulb‐derived fibroblast‐like cells expressed CD29, CD44, CD90, CD105, CD166 but not CD34 and CD45, consistent with the characteristics of MSCs. Long cylindical cells that spontaneously contracted were only observed following 7 days of co‐culture of MSCs with rat cardiomyocytes plus neuregulin‐1, bFGF and forskolin. RT–PCR and western blot analysis indicated that the cylindrical cells expressed myocardial markers, such as Nkx2.5, GATA4, sarcomeric α‐actinin, cardiac troponin I, cardiac myosin heavy chain, atrial natriuretic peptide and connexin 43. They also contained sarcomeres and gap junction and were sensitive to pharmacological treatments (adrenal and cholinergic agonists and antagonists). These findings indicate that rat olfactory bulb‐derived fibroblast‐like cells with MSC characteristics can differentiate into myocardial‐like cells. Copyright

Baicalein increases keratin 1 and 10 expression in HaCaT keratinocytes via TRPV4 receptor activation

In this study, we characterized the effect of baicalein on the regulation of keratinocyte differentiation and proliferation, which are abnormal in atopic dermatitis or psoriasis. Treatment of HaCaT keratinocytes with 10 μm baicalein slightly inhibited cell growth, caused morphological differentiation and increased expression of keratins 1 and 10 (K1/K10) without affecting ROS generation, cytochrome c release or apoptosis. Baicalein treatment caused growth arrest in G0/G1 phase and also induced Ca2+ influx via TRPV4 receptor activation. Phosphorylation of ERK, Akt and p38 MAPK, but not JNK, was increased by baicalein, and inhibition of phosphorylation of ERK, but not that of Akt or p38 MAPK, blocked the baicalein‐induced increase in K1/K10 expression, suggesting that ERK activation is involved in this increase. Removal of extracellular Ca2+ or blockade of Ca2+ influx by pharmacological inhibition or silencing of the TRPV4 receptor did not affect growth arrest, ROS generation or apoptosis, but inhibited baicalein‐induced ERK phosphorylation and K1/K10 expression. Thus, baicalein treatment increases differentiation, and decreases proliferation, of keratinocytes. The mechanism of differentiation of keratinocytes is distinct from that of proliferation, the former being Ca2+ dependent and the latter Ca2+ independent.

Phosphorylation promotes the desensitization of the opioid-induced Ca2+ increase in NG108-15 cells.

Using the fluorescent Ca2+ indicator fura-2, we demonstrated that, in a single NG108-15 cell, acute repetitive challenge with leucine-enkephalin (EK) results in a gradual reduction of the increase of the cytosolic Ca2+ concentration ([Ca2+]i) at agonist exposure times of 90 s or less; increasing the EK exposure time of each challenge from 30 to 90 s results in greater desensitization, with complete desensitization occurring at 90 s exposure. Similar results are seen with ATP. In opioid-desensitized cells, bradykinin can still induce a marked [Ca2+]i increase, while exposure of desensitized cell to 50 mM K+ restores the response EK-induced, suggesting a role of intracellular Ca2+ stores in the desensitization process. Pretreatment of cells with certain protein kinase inhibitors, including N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004) and staurosporine, prevented desensitization, while others, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and {1-[N, O-bis-(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenyl-piperazine (KN-62), had no effect. In contrast, activation of protein kinase C by phorbol 12-myristate 13-acetate promoted desensitization. Thus, the desensitization is dependent on protein phosphorylation. HA1004 alone did not alter EK- or bradykinin-induced inositol 1,4, 5-trisphosphate (IP3) generation; however, the inhibitory effect of calyculin A on EK- or bradykinin-induced IP3 generation was reversed by HA1004. In addition, in the presence of HA1004, the blockade of Ca2+ influx by either verapamil or removal of extracellular Ca2+ or the depletion of Ca2+ pools by thapsigargin still led to desensitization, suggesting that phosphorylation does not alter the activity of the Ca2+ transporters involved in Ca2+ influx and Ca2+ release. Our results imply that emptying of intracellular Ca2+ stores and protein phosphorylation in the phospholipase C signaling pathway play roles in the process of desensitization.