Conjugated linoleic acid induces human adipocyte delipidation: autocrine/paracrine regulation of MEK/ERK signaling by adipocytokines

Dietary conjugated linoleic acid (CLA) reduces body fat in animals and some humans. Here we show that trans-10, cis-12 CLA, but not cis-9, trans-11 CLA, when added to cultures of stromal vascular cells containing newly differentiated human adipocytes, caused a time-dependent decrease in triglyceride content, insulin-stimulated glucose and fatty acid uptake, incorporation into lipid, and oxidation compared with controls. In parallel, gene expression of peroxisome proliferator-activated receptor-gamma and many of its downstream targets were diminished by trans-10, cis-12 CLA, whereas leptin gene expression was increased. Prior to changes in gene expression and metabolism, trans-10, cis-12 CLA caused a robust and sustained activation of mitogen-activated protein kinase kinase/extracellular signal-related kinase (MEK/ERK) signaling. Furthermore, the trans-10, cis-12 CLA-mediated activation of MEK/ERK could be attenuated by pretreatment with U0126 and pertussis toxin. In parallel, pretreatment with U0126 blocked the ability of trans-10, cis-12 CLA to alter gene expression and attenuate glucose and fatty acid uptake of the cultures. Intriguingly, the induction by CLA of MEK/ERK signaling was linked to hypersecretion of adipocytokines interleukin-6 and interleukin-8. Collectively, these data demonstrate for the first time that trans-10, cis-12 CLA decreases the triglyceride content of newly differentiated human adipocytes by inducing MEK/ERK signaling through the autocrine/paracrine actions of interleukins-6 and 8.     

Laminin-1 induces neurite outgrowth in human mesenchymal stem cells in serum/differentiation factors-free conditions through activation of FAK-MEK/ERK signaling pathways

Mesenchymal stem cells (MSCs) can be differentiated into cell types derived from all three germ layers by manipulating culture conditions in vitro. A multitude of growth and differentiation factors have been employed for driving MSCs towards a neuronal phenotype. In the present study, we investigated the potential of extracellular matrix (ECM) proteins—fibronectin, collagen-1, collagen-IV, laminin-1, and laminin-10/11, to induce a neuronal phenotype in bone marrow derived human MSCs in the absence of growth factors/differentiating agents. All of the ECM proteins tested were found to support adhesion of MSCs to different extents. However, direct interaction only with laminin-1 triggered sprouting of neurite-like processes. Cells plated on laminin-1 exhibited neurite out growth as early as 3 h, and by 24 h, the cells developed elaborate neurites with contracted cell bodies and neuronal-like morphology. Function-blocking antibodies directed against α6 and β1 integrin subunits inhibited neurite formation on laminin-1 which confirmed the involvement of integrin α6β1 in neurite outgrowth. Mechanistic studies revealed that cell adhesion to laminin-1 activated focal adhesion kinase (FAK), and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways. Abrogation of FAK phosphorylation by herbimycin-A inhibited neurite formation and also decreased activities of MEK and ERK. Pharmacological inhibitors of MEK (U0126) and ERK (PD98059) also blocked neurite outgrowth in cells plated on laminin-1. Our study demonstrates the involvement of integrin α6β1 and FAK-MEK/ERK signaling pathways in laminin-1-induced neurite outgrowth in MSCs in the absence of serum and differentiation factors.     

Hyaluronan substratum induces multidrug resistance in human mesenchymal stem cells via CD44 signaling

Little information is available concerning multidrug resistance (MDR) in mesenchymal stem cells, although several studies have reported that MDR is associated with hyaluronan in neoplastic cells. We have evaluated whether a hyaluronan-coated surface modulates MDR in placenta-derived human mesenchymal stem cells (PDMSCs). We have found that PDMSCs cultured on a tissue-culture polystyrene surface coated with 30 μg/cm² hyaluronan are more resistant than control PDMSCs to doxorubicin. Inhibiting PI3K/Akt signaling has shown that the PI3K/Akt pathway modulates both P-glycoprotein activity and doxorubicin resistance. In addition, 10 μM verapamil dramatically suppresses the doxorubicin resistance induced by the hyaluronan-coated surface, indicating that P-glycoprotein activity is necessary for MDR. We have further found that PDMSCs treated with CD44 small interfering RNA (siRNA) and grown on a polystyrene surface coated with 30 μg/cm² hyaluronan have fewer P-glycoprotein⁺ cells and lower CD44 expression levels (less than 60% in both cases) than PDMSCs not treated with CD44 siRNA and grown on the hyaluronan-coated surface. Moreover, treatment with CD44 siRNA suppresses the hyaluronan-substratum-induced doxorubicin resistance. We conclude that a hyaluronan substratum induces MDR in PDMSCs through CD44 signaling. This work was supported by research grant NSC95–2745-B-006–003-MY2 from the National Science Council, Taiwan, and by Landmark Project Grant A25, funded by the Taiwan Ministry of Education, from National Cheng Kung University.     

Oxidative stress induces senescence in human mesenchymal stem cells

Mesenchymal stem cells (MSCs) contribute to tissue repair in vivo and form an attractive cell source for tissue engineering. Their regenerative potential is impaired by cellular senescence. The effects of oxidative stress on MSCs are still unknown. Our studies were to investigate into the proliferation potential, cytological features and the telomere linked stress response system of MSCs, subject to acute or prolonged oxidant challenge with hydrogen peroxide. Telomere length was measured using the telomere restriction fragment assay, gene expression was determined by rtPCR. Sub-lethal doses of oxidative stress reduced proliferation rates and induced senescent-morphological features and senescence-associated β-galactosidase positivity. Prolonged low dose treatment with hydrogen peroxide had no effects on cell proliferation or morphology. Sub-lethal and prolonged low doses of oxidative stress considerably accelerated telomere attrition. Following acute oxidant insult p21 was up-regulated prior to returning to initial levels. TRF1 was significantly reduced, TRF2 showed a slight up-regulation. SIRT1 and XRCC5 were up-regulated after oxidant insult and expression levels increased in aging cells. Compared to fibroblasts and chondrocytes, MSCs showed an increased tolerance to oxidative stress regarding proliferation, telomere biology and gene expression with an impaired stress tolerance in aged cells.     

NOS inhibition synchronizes calcium oscillations in human adipose tissue-derived mesenchymal stem cells by increasing gap-junctional coupling

Adipose tissue-derived mesenchymal stem cells (ASCs) are a promising stem cell source for cell transplantation. We demonstrate that undifferentiated ASCs display robust oscillations of intracellular calcium [Ca(2+) ](i) which may be associated with stem cell maintenance since oscillations were absent in endothelial cell differentiation medium supplemented with FGF-2. [Ca(2+) ](i) oscillations were dependent on extracellular Ca(2+) and Ca(2+) release from intracellular stores since they were abolished in Ca(2+) -free medium and in the presence of the store-depleting agent thapsigargin. They were inhibited by the phospholipase C antagonist U73,122, the inositol 1,4,5-trisphosphate (InsP(3) ) receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) as well as by the gap-junction uncouplers 1-heptanol and carbenoxolone, indicating regulation by the InsP(3) pathway and dependence on gap-junctional coupling. Cells endogenously generated nitric oxide (NO), expressed NO synthase 1 (NOS 1) and connexin 43 (Cx 43). The nitric oxide NOS inhibitors NG-monomethyl-L-arginine (L-NMMA), N(G)-nitro-L-arginine methyl ester (L-NAME), 2-ethyl-2-thiopseudourea, and diphenylene iodonium as well as si-RNA-mediated down-regulation of NOS 1 synchronized [Ca(2+) ](i) oscillations between individual cells, whereas the NO-donors S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) as well as the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) were without effects. The synchronization of [Ca(2+) ](i) oscillations was due to an improvement of intracellular coupling since fluorescence recovery after photobleaching (FRAP) revealed increased reflow of fluorescent calcein into the bleached area in the presence of the NOS inhibitors DPI and L-NAME. In summary our data demonstrate that intracellular NO levels regulate synchronization of [Ca(2+) ](i) oscillations in undifferentiated ASCs by controlling gap-junctional coupling.     

Calcium-sensing receptor activation induces intracellular calcium oscillations

Parathyroid hormone secretion isexquisitely sensitive to small changes in serum Ca²⁺concentration, and these responses are transduced via theCa²⁺-sensing receptor (CaR). We utilized heterologousexpression in HEK-293 cells to determine the effects of small,physiologically relevant perturbations in extracellularCa²⁺ on CaR signaling viaphosphatidylinositol-phospholipase C, using changes in fura 2 fluorescence to quantify intracellular Ca²⁺. Chronicexposure of CaR-transfected cells to Ca²⁺ in the range from0.5 to 3 mM modulated the resting intracellular Ca²⁺concentration and the subsequent cellular responses to acute extracellular Ca²⁺ perturbations but had no effect onthapsigargin-sensitive Ca²⁺ stores. Modest,physiologically relevant increases in extracellular Ca²⁺concentration (0.5 mM increments) caused sustained (30-40 min) low-frequency oscillations of intracellular Ca²⁺ (~45 speak to peak interval). Oscillations were eliminated by 1 µMthapsigargin but were insensitive to protein kinase inhibitors (staurosporine, KN-93, or bisindolylmaleimide I). Staurosporine didincrease the fraction of cells oscillating at a given extracellular Ca²⁺ concentration. Serum Ca²⁺ concentrationsthus chronically regulate cells expressing CaR, and small perturbationsin extracellular Ca²⁺ alter both resting intracellularCa²⁺ as well as Ca²⁺ dynamics.

     

Bladder cancer cell in co-culture induces human stem cell differentiation to urothelial cells through paracrine FGF10 signaling

Fibroblast growth factor 10 (FGF10) is required for embryonic epidermal morphogenesis including brain development, lung morphogenesis, and initiation of limb bud formation. In this study, we investigated the role of FGF10 as a lead induction factor for stem cell differentiation toward urothelial cell. To this end, human multipotent stem cell in vitro system was employed. Human amniotic fluid stem cells were co-cultured with immortalized bladder cancer lines to induce directed differentiation into urothelial cells. Urothelial markers, uroplakin II, III, and cytokeratin 8, were monitored by RT-PCR, immunocytochemistry, and Western blot analysis. Co-cultured stem cells began to express uroplakin II, III, and cytokeratin 8. Targeted FGF10 gene knockdown from bladder cancer cells abolished the directed differentiation. In addition, when FGF10 downstream signaling was blocked with the Mek inhibitor, the co-culture system lost the capacity to induce urothelial differentiation. Exogenous addition of recombinant FGF10 protein promoted stem cell differentiation into urothelium cell lineage. Together, this report suggests that paracrine FGF10 signaling stimulates the differentiation of human stem cell into urothelial cells. Current study provides insight into the potential role of FGF10 as a lead growth factor for bladder regeneration and its therapeutic application for bladder transplantation.     

Sphingosylphosphorylcholine induces proliferation of human adipose tissue-derived mesenchymal stem cells via activation of JNK

Sphingosylphosphorylcholine (SPC) has been implicated in a variety of cellular responses, including proliferation and differentiation. In this study, we demonstrate that d-erythro-SPC, but not l-threo-SPC, stereoselectively stimulated the proliferation of human adipose tissue-derived mesenchymal stem cells (hADSCs), with a maximal increase at 5 microM, and increased the intracellular concentration of Ca(2+) ([Ca(2+)](i)) in hADSCs, which do not express known SPC receptors (i.e., OGR1, GPR4, G2A, and GPR12). The SPC-induced proliferation and increase in [Ca(2+)](i) were sensitive to pertussis toxin (PTX) and the phospholipase C (PLC) inhibitor U73122, suggesting that PTX-sensitive G proteins, Gi or Go, and PLC are involved in SPC-induced proliferation. In addition, SPC treatment induced the phosphorylation of c-Jun and extracellular signal-regulated kinase, and SPC-induced proliferation was completely prevented by pretreatment with the c-Jun N-terminal kinase (JNK)-specific inhibitor SP600125 but not with the MEK-specific inhibitor U0126. Furthermore, the SPC-induced proliferation and JNK activation were completely attenuated by overexpression of a dominant negative mutant of JNK2, and the SPC-induced activation of JNK was inhibited by pretreatment with PTX or U73122. Treatment of hADSCs with lysophosphatidic acid (LPA) receptor antagonist, Ki16425, had no impact on the SPC-induced increase in [Ca(2+)](i). However, SPC-induced proliferation was partially, but significantly, attenuated by pretreatment of the cells with Ki16425.These results indicate that SPC stimulates the proliferation of hADSCs through the Gi/Go-PLC-JNK pathway and that LPA receptors may be responsible in part for the SPC-induced proliferation.     

Reactive oxygen species alter autocrine and paracrine signaling

Cytochrome P450 (P450) 3A4 (CYP3A4) is the most abundant P450 protein in human liver and intestine and is highly inducible by a variety of drugs and other compounds. The P450 catalytic cycle is known to uncouple and release reactive oxygen species (ROS), but the effects of ROS from P450 and other enzymes in the endoplasmic reticulum have been poorly studied from the perspective of effects on cell biology. In this study, we expressed low levels of CYP3A4 in HepG2 cells, a human hepatocarcinoma cell line, and examined effects on intracellular levels of ROS and on the secretion of a variety of growth factors that are important in extracellular communication. Using the redox-sensitive dye RedoxSensor red, we demonstrate that CYP3A4 expression increases levels of ROS in viable cells. A custom ELISA microarray platform was employed to demonstrate that expression of CYP3A4 increased secretion of amphiregulin, intracellular adhesion molecule 1, matrix metalloprotease 2, platelet-derived growth factor (PDGF), and vascular endothelial growth factor, but suppressed secretion of CD14. The antioxidant N-acetylcysteine suppressed all P450-dependent changes in protein secretion except for CD14. Quantitative RT-PCR demonstrated that changes in protein secretion were consistently associated with corresponding changes in gene expression. Inhibition of the NF-κB pathway blocked P450 effects on PDGF secretion. CYP3A4 expression also altered protein secretion in human mammary epithelial cells and C10 mouse lung cells. Overall, these results suggest that increased ROS production in the endoplasmic reticulum alters the secretion of proteins that have key roles in paracrine and autocrine signaling.     

Endocrine and paracrine calcium signaling in bile duct cells.

Bile duct cells play an important role in maintaining, modifying and augmenting bile flow. It is well established that cyclic AMP (cAMP) is an important second messenger for secretion in these cells, but less is known about cytosolic Ca2+ (Ca2+i). Here we review evidence that ATP and acetylcholine (ACh) are Ca2+i agonists for bile duct cells, and that these agonists increase Ca2+i through inositol 1,4,5-trisphosphate (InsP3). We also review data suggesting that hepatocytes have the ability to secrete ATP, so that they may serve as a paracrine source for this signaling molecule in vivo. Finally, we compare the effects of cAMP and Ca2+i on secretion, both in isolated bile duct units and isolated hepatocyte couplets. Implications and future directions for studying the role of Ca2+i in bile ductular secretion are discussed.     

Intracellular signaling following myocardial stretch: an autocrine/paracrine loop

The stretch of adult papillary muscle elicits a chain of autocrine/paracrine events in which the Na(+)/H(+) exchanger (NHE-1) activation is the central step. This activation is induced by a sequential angiotensin II-endothelin (Ang II-ET) release and results in an increase in intracellular Na(+) (Na(+)(i)) without significant changes in intracellular pH. The increase in Na(+)(i) negatively shifts the reverse potential of the Na(+)/Ca(2+) exchanger (NCX) thus inducing cell Ca(2+) influx that augments myocardial contractility. This increase in force represents the mechanical counterpart of the autocrine/paracrine mechanism triggered by stretch and has been called the slow force response (SFR) to stretch.     

A role for the primary cilium in paracrine signaling between mechanically stimulated osteocytes and mesenchymal stem cells

Bone turnover is a mechanically regulated process, coordinated in part by the network of mechanosensitive osteocytes residing within the tissue. The recruitment and bone forming activity of the mesenchymal derived osteoblast is determined by numerous factors including mechanical loading. It is therefore somewhat surprising that although mechanically regulated signaling between the coordinating osteocytes and mesenchymal stem cells (MSCs) should exist, to date it has not been directly demonstrated. In this study, conditioned media from mechanically stimulated osteocytes (MLO-Y4 cell line) was collected and added to MSCs (C3H10T1/2 cell line). The addition of mechanically stimulated osteocyte conditioned media resulted in a significant upregulation of the osteogenic genes OPN and COX-2 in MSCs compared to statically cultured conditioned media, demonstrating a novel paracrine signaling mechanism between the two cell types. The same mechanically conditioned media did not alter gene expression in osteoblasts (MC3T3 cell line), and mechanically stimulated osteoblast conditioned media did not alter gene expression in MSCs demonstrating that this signaling is unique to osteocytes and MSCs. Finally, the upregulation in osteogenic genes in MSCs was not observed if primary cilia formation was inhibited prior to mechanical stimulation of the osteocyte. In summary, the results of this study indicate that soluble factors secreted by osteocytes in response to mechanical stimulation can enhance osteogenic gene expression in MSCs demonstrating a novel, unique signaling mechanism and introduces a role for the primary cilium in flow mediated paracrine signaling in bone thereby highlighting the cilium as a potential target for therapeutics aimed at enhancing bone formation.     

MAP kinase and calcium signaling mediate fluid flow-induced human mesenchymal stem cell proliferation

Mechanical signals are important regulators of skeletal homeostasis, and strain-induced oscillatory fluid flow is a potent mechanical stimulus. Although the mechanisms by which osteoblasts and osteocytes respond to fluid flow are being elucidated, little is known about the mechanisms by which bone marrow-derived mesenchymal stem cells respond to such stimuli. Here we show that the intracellular signaling cascades activated in human mesenchymal stem cells by fluid flow are similar to those activated in osteoblastic cells. Oscillatory fluid flow inducing shear stresses of 5, 10, and 20 dyn/cm² triggered rapid, flow rate-dependent increases in intracellular calcium that pharmacological studies suggest are inositol trisphosphate mediated. The application of fluid flow also induced the phosphorylation of extracellular signal-regulated kinase-1 and -2 as well as the activation of the calcium-sensitive protein phosphatase calcineurin in mesenchymal stem cells. Activation of these signaling pathways combined to induce a robust increase in cellular proliferation. These data suggest that mechanically induced fluid flow regulates not only osteoblastic behavior but also that of mesenchymal precursors, implying that the observed osteogenic response to mechanical loading may be mediated by alterations in the cellular behavior of multiple members of the osteoblast lineage, perhaps by a common signaling pathway. mechanotransduction; bone; marrow     

Hedgehog-mediated paracrine interaction between hepatic stellate cells and marrow-derived mesenchymal stem cells

During liver injury, bone marrow-derived mesenchymal stem cells (MSCs) can migrate and differentiate into hepatocytes. Hepatic stellate cell (SC) activation is a pivotal event in the development of liver fibrosis. Therefore, we hypothesized that SCs may play an important role in regulating MSC proliferation and differentiation through the paracrine signaling pathway. We demonstrate that MSCs and SCs both express hedgehog (Hh) pathway components, including its ligands, receptors, and target genes. Transwell co-cultures of SCs and MSCs showed that the SCs produced sonic hedgehog (Shh), which enhanced the proliferation and differentiation of MSCs. These findings demonstrate that SCs indirectly modulate the activity of MSCs in vitro via the Hh pathway, and provide a plausible explanation for the mechanisms of transplanted MSCs in the treatment of liver fibrosis.     

Endometriosis stromal cells induce bone marrow mesenchymal stem cell differentiation and PD-1 expression through paracrine signaling

Molecular and Cellular Biochemistry - Endometriosis is an estrogen-dependent, inflammatory gynecological disorder characterized by the growth of endometrial cells in lesions outside the uterus....     

Extracellular NAD(+) induces calcium signaling and apoptosis in human osteoblastic cells

ADP-ribosyl cyclase/CD38 is a bifunctional enzyme that catalyzes at its ectocellular domain the synthesis from NAD(+) (cyclase) and the hydrolysis (hydrolase) of the calcium-mobilizing second messenger cyclic ADP ribose (cADPR). Furthermore, CD38 mediates cADPR influx inside a number of cells, thereby inducing Ca(2+) mobilization. Intracellularly, cADPR releases Ca(2+) from ryanodine-sensitive pools, thus activating several Ca(2+)-dependent functions. Among these, the inhibition of osteoclastic-mediated bone resorption has been demonstrated. We found that HOBIT human osteoblastic cells display ADP-ribosyl cyclase activity and we examined the effects of CD38 stimulation on osteoblasts function. Extracellular NAD(+) induced elevation of cytosolic calcium due to both Ca(2+) influx from the extracellular medium and Ca(2+) release from ryanodine-sensitive intracellular stores. Culturing these cells in the presence of NAD(+) caused a complete growth arrest with a time-dependent decrease of cell number and the appearance of apoptotic nuclei. The first changes could be observed after 24 h of treatment and became fully evident after 72-96 h. We propose a role of extracellular NAD(+) in bone homeostatic control.