Induction of Melanogenesis by Tetradecanoylphorbol‐13 Acetate and Endothelin 3 in Embryonic Avian Peripheral Nerve Cultures

In this study, we have analyzed the melanogenic potential of Schwann cells using in vitro cell cultures of embryonic quail peripheral nerves. It is shown that in Schwann cells, two factors, 12‐O‐tetradecanoylphorbol‐13 acetate (TPA) and endothelin 3, trigger a differentiation pathway toward melanocytes, and that Steel factor has no effect on these cells unless treated simultaneously with TPA. In these cultures, TPA induces the expression of c‐kit, whereas Steel factor enhances the development of melanocytes. In the assay system we employed, neither neuronal nor catecholaminergic phenotypes were obtained, regardless of various combinations of related factors added to the culture medium. These data support our previous observations indicating the existence of bipotent progenitors that are capable of differentiating into Schwann cells or into melanocytes, and the regulatory role of endothelin 3 on those precursors, as revealed by the clonal culture of neural crest cells.     

Role of sialic acid‐containing glycans of matrix metalloproteinase‐9 (MMP‐9) in the interaction between MMP‐9 and staphylococcal superantigen‐like protein 5

Staphylococcal superantigen‐like proteins (SSL) show no superantigenic activity but have recently been considered to act as immune suppressors. It was previously reported that SSL5 bound to P‐selectin glycoprotein ligand‐1 (PSGL‐1) and matrix metalloproteinase (MMP)‐9, leading to inhibition of leukocyte adhesion and invasion. These interactions were suggested to depend on sialic acid‐containing glycans of MMP‐9, but the roles of sialic acids in the interaction between SSL5 and MMP‐9 are still controversial. In the present study, we prepared recombinant glutathione S‐transferase‐tagged SSL5 (GST‐SSL5) and analyzed its binding capacity to MMP‐9 by pull‐down assay after various modifications of its carbohydrate moieties. We observed that GST‐SSL5 specifically bound to MMP‐9 from a human monocytic leukemia cell line (THP‐1 cells) and inhibited its enzymatic activity in a concentration‐dependent manner. After MMP‐9 was treated with neuraminidase, its binding activity towards GST‐SSL5 was markedly decreased. Furthermore, recombinant MMP‐9 produced by sialic acid‐deficient Lec2 mutant cells showed much lower affinity for SSL5 than that produced by wild‐type CHO‐K1 cells. Treatment of MMP‐9 with PNGase F to remove N‐glycan resulted in no significant change in the GST‐SSL5/MMP‐9 interaction. In contrast, the binding of GST‐SSL5 to MMP‐9 secreted from THP‐1 cells cultured in the presence of an inhibitor for the biosynthesis of O‐glycan (benzyl‐GalNAc) was weaker than the binding of GST‐SSL5 to MMP‐9 secreted from untreated cells. These results strongly suggest the importance of the sialic acid‐containing O‐glycans of MMP‐9 for the interaction of MMP‐9 with GST‐SSL5.

     

Aromatic‐turmerone attenuates invasion and expression of MMP‐9 and COX‐2 through inhibition of NF‐κB activation in TPA‐induced breast cancer cells

Recent evidence suggests that breast cancer is one of the most common forms of malignancy in females, and metastasis from the primary cancer site is the main cause of death. Aromatic (ar)‐turmerone is present in Curcuma longa and is a common remedy and food. In the present study, we investigated the inhibitory effects of ar‐turmerone on expression and enzymatic activity levels of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced matrix metalloproteinase (MMP)‐9 and cyclooxygenaase‐2 (COX‐2) in breast cancer cells. Our data indicated that ar‐turmerone treatment significantly inhibited enzymatic activity and expression of MMP‐9 and COX‐2 at non‐cytotoxic concentrations. However, the expression of tissue inhibitor of metalloproteinase (TIMP)‐1, TIMP‐2, MMP‐2, and COX‐1 did not change upon ar‐turmerone treatment. We found that ar‐turmerone inhibited the activation of NF‐κB, whereas it did not affect AP‐1 activation. Moreover, The ChIP assay revealed that in vivo binding activities of NF‐κB to the MMP‐9 and COX‐2 promoter were significantly inhibited by ar‐turmerone. Our data showed that ar‐turmerone reduced the phosphorylation of PI3K/Akt and ERK1/2 signaling, whereas it did not affect phosphorylation of JNK or p38 MAPK. Thus, transfection of breast cancer cells with PI3K/Akt and ERK1/2 siRNAs significantly decreased TPA‐induced MMP‐9 and COX‐2 expression. These results suggest that ar‐turmerone suppressed the TPA‐induced up‐regulation of MMP‐9 and COX‐2 expression by blocking NF‐κB, PI3K/Akt, and ERK1/2 signaling in human breast cancer cells. Furthermore, ar‐turmerone significantly inhibited TPA‐induced invasion, migration, and colony formation in human breast cancer cells. J. Cell. Biochem. 113: 3653–3662, 2012. © 2012 Wiley Periodicals, Inc.     

Tumor-Promoting Phorbol Esters Promote Melanogenesis and Prevent Expression of the Adrenergic Phenotype in Quail Neural Crest Cells

Tumor-promoting phorbol esters were used to manipulate the in vitro development of neural crest cells. When plated at clonal density in secondary culture, quail neural crest cells from the trunk region gave rise to three types of colonies, pigmented, unpigmented, and mixed. Pigmented colonies consisted exclusively of melanocytes; up to 50% of the unpigmented and mixed colonies contained adrenergic nerve cells which could be identified by a catecholamine-specific histofluorescence method. Addition of potent tumor promoters to the culture medium shortened the doubling time of neural crest cells and altered their morphologic appearance. It also delayed the onset of pigmentation, prevented the expression of the adrenergic phenotype, reduced the number of unpigmented and mixed colonies, and increased the number of pigmented colonies, most likely by directing progenitor cells preferentially to the melanogenic pathway. There was a clear correlation between the ability of phorbol esters to promote skin tumors in mice and their ability to interfere with the in vitro development of quail neural crest cells. The potent promoters 12–0–tetradecanoyl phorbol 13–acetate (TPA) and phorbol 12,13–didecanoate (PDD) were most effective, phorbol 12,13–diacetate (PDA) was considerably less effective, the nonpromoting analogues 4–0–methyl 12–0–tetradecanoyl phorbol 13–acetate (4–0–Me-TPA) and 4α-phorbol 12,13–didecanoate (4α-PDD) and the parent alcohol phorbol (PHR) had little or no effect.     

Nitric oxide‐mediated regulation of ‐amyloid clearance via alterations of MMP‐9/TIMP‐1

Fibrillar amyloid plaques are largely composed of amyloid‐beta (Aβ) peptides that are metabolized into products, including Aβ1‐16, by proteases including matrix metalloproteinase 9 (MMP‐9). The balance between production and degradation of Aβ proteins is critical to amyloid accumulation and resulting disease. Regulation of MMP‐9 and its endogenous inhibitor tissue inhibitor of metalloproteinase (TIMP)‐1 by nitric oxide (NO) has been shown. We hypothesize that nitric oxide synthase (NOS2) protects against Alzheimer's disease pathology by increasing amyloid clearance through NO regulation of MMP‐9/TIMP‐1 balance. We show NO‐mediated increased MMP‐9/TIMP‐1 ratios enhanced the degradation of fibrillar Aβ in vitro, which was abolished when silenced for MMP‐9 protein translation. The in vivo relationship between MMP‐9, NO and Aβ degradation was examined by comparing an Alzheimer's disease mouse model that expresses NOS2 with a model lacking NOS2. To quantitate MMP‐9 mediated changes, we generated an antibody recognizing the Aβ1‐16 fragment, and used mass spectrometry multi‐reaction monitoring assay for detection of immunoprecipitated Aβ1‐16 peptides. Aβ1‐16 levels decreased in brain lysates lacking NOS2 when compared with strains that express human amyloid precursor protein on the NOS2 background. TIMP‐1 increased in the APPSwDI/NOS2^?/? mice with decreased MMP activity and increased amyloid burden, thereby supporting roles for NO in the regulation of MMP/TIMP balance and plaque clearance.     

Impact of 4‐hydroxynonenal on matrix metalloproteinase‐9 regulation in lipopolysaccharide‐stimulated RAW 264.7 cells

Tissue degradation and leukocyte extravasation suggest proteolytic destruction of the extracellular matrix (ECM) during severe malaria. Matrix metalloproteinases (MMPs) play an established role in ECM turnover, and increased MMP‐9 protein abundance is correlated with malarial infection. The malaria pigment hemozoin (Hz) is a heme detoxification biomineral that is produced during infection and associated with biologically active lipid peroxidation products such as 4‐hydroxynonenal (HNE) adsorbed to its surface. Hz has innate immunomodulatory activity, and many of its effects can be reproduced by exogenously added HNE. Hz phagocytosis enhances MMP‐9 expression in monocytes; thus, this study was designed to examine the ability of HNE to alter MMP‐9 regulation in activated cells of macrophage lineage. Data show that treatment of lipopolysaccharide‐stimulated RAW 264.7 cells with HNE increased MMP‐9 secretion and activity. HNE treatment abolished the cognate tissue inhibitor of metalloproteinase‐1 protein levels, further decreasing MMP‐9 regulation. Phosphorylation of both p38 mitogen‐activated protein kinase (MAPK) and c‐Jun NH2‐terminal kinase was induced by HNE, but only p38 MAPK inhibition lessened MMP‐9 secretion. These results demonstrate the in vitro ability of HNE to cause MMP‐9 dysregulation in an activated cell model. The findings may extend to myriad pathologies associated with lipid peroxidation and elevated MMP‐9 levels leading to tissue damage. Copyright © 2015 John Wiley & Sons, Ltd.     

Caveolin‐1 mediates tissue plasminogen activator‐induced MMP‐9 up‐regulation in cultured brain microvascular endothelial cells

Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase‐9 (MMP‐9) activity in the ischemic brain, which exacerbates blood‐brain barrier injury and increases the risk of symptomatic cerebral hemorrhage. The mechanism through which tPA enhances MMP‐9 activity is not well understood. Here we report an important role of caveolin‐1 in mediating tPA‐induced MMP‐9 synthesis. Brain microvascular endothelial cell line bEnd3 cells were incubated with 5 or 20 μg/ml tPA for 24 hrs before analyzing MMP‐9 levels in the conditioned media and cellular extracts by gelatin zymography. tPA at a dose of 20 μg/mL tPA, but not 5 μg/mL, significantly increased MMP‐9 level in cultured media while decreasing it in cellular extracts. Concurrently, tPA treatment induced a 2.3‐fold increase of caveolin‐1 protein levels in endothelial cells. Interestingly, knockdown of Cav‐1 with siRNA inhibited tPA‐induced MMP‐9 mRNA up‐regulation and MMP‐9 increase in the conditioned media, but did not affect MMP‐9 decrease in cellular extracts. These results suggest that caveolin‐1 critically contributes to tPA‐mediated MMP‐9 up‐regulation, but may not facilitate MMP‐9 secretion in endothelial cells.

     

MMP‐13 deletion decreases profibrogenic molecules and attenuates N‐nitrosodimethylamine‐induced liver injury and fibrosis in mice

Connective tissue growth factor (CTGF) is involved in inflammation, pathogenesis and progression of liver fibrosis. Matrix metalloproteinase‐13 (MMP‐13) cleaves CTGF and releases several fragments, which are more potent than the parent molecule to induce fibrosis. The current study was aimed to elucidate the significance of MMP‐13 and CTGF and their downstream effects in liver injury and fibrosis. Hepatic fibrosis was induced using intraperitoneal injections of N‐nitrosodimethylamine (NDMA) in doses of 10 μg/g body weight on three consecutive days of each week over a period of 4 weeks in both wild‐type (WT) and MMP‐13 knockout mice. Administration of NDMA resulted in marked elevation of AST, ALT, TGF‐β1 and hyaluronic acid in the serum and activation of stellate cells, massive necrosis, deposition of collagen fibres and increase in total collagen in the liver of WT mice with a significant decrease in MMP‐13 knockout mice. Protein and mRNA levels of CTGF, TGF‐β1, α‐SMA and type I collagen and the levels of MMP‐2, MMP‐9 and cleaved products of CTGF were markedly increased in NDMA‐treated WT mice compared to the MMP‐13 knockout mice. Blocking of MMP‐13 with CL‐82198 in hepatic stellate cell cultures resulted in marked decrease of the staining intensity of CTGF as well as protein levels of full‐length CTGF and its C‐terminal fragments and active TGF‐β1. The data demonstrate that MMP‐13 and CTGF play a crucial role in modulation of fibrogenic mediators and promote hepatic fibrogenesis. Furthermore, the study suggests that blocking of MMP‐13 and CTGF has potential therapeutic implications to arrest liver fibrosis.     

MMP‐2 and MMP‐13 affect vasculogenic mimicry formation in large cell lung cancer

Matrix metalloproteinases (MMPs) have critical functions in tumour vasculogenic mimicry (VM). This study explored the mechanisms underlying MMP‐13 and MMP‐2 regulation of tumour VM formation in large cell lung cancer (LCLC). In our study, laminin5 (Ln‐5) fragments cleaved by MMP‐2 promoted tubular structure formation by the LCLC cell lines H460 and H661 in three‐dimensional (3D) cultures. Transient up‐regulation of MMP‐13 or treatment with recombinant MMP‐13 protein abrogated tubular structure formation of H460 cells in 3D culture. Treated cells with Ln‐5 fragments cleaved by MMP‐2 stimulated EGFR and F‐actin expression. Ln‐5 fragments cleaved by MMP‐13 decreased EGFR/F‐actin expression and disrupted VM formation. MMP‐13 expression was negatively correlated with VM, Ln‐5 and EGFR in LCLC tissues and xenograft. In vivo experiments revealed that VM was decreased when the number of endothelium‐dependent vessels (EDVs) increased during xenograft tumour growth, whereas MMP‐13 expression was progressively increased. In conclusion, MMP‐2 promoted and MMP‐13 disrupted VM formation in LCLC by cleaving Ln‐5 to influence EGFR signal activation. MMP‐13 may regulate VM and EDV formation.     

Involvement of p38 MAPK in haemozoin‐dependent MMP‐9 enhancement in human monocytes

The lipid moiety of natural haemozoin (nHZ, malarial pigment) was previously shown to enhance expression and release of human monocyte matrix metalloproteinase‐9 (MMP‐9), and a major role for 15‐(S,R)‐hydroxy‐6,8,11,13‐eicosatetraenoic acid (15‐HETE), a nHZ lipoperoxidation product, was proposed. Here, the underlying mechanisms were investigated, focusing on the involvement of mitogen‐activated protein kinases (MAPKs). Results showed that nHZ promoted either early or late p38 MAPK phosphorylation; however, nHZ did not modify basal phosphorylation/expression ratios of extracellular signal‐regulated kinase‐1/2 and c‐jun N‐terminal kinase‐1/2. 15‐HETE mimicked nHZ effects on p38 MAPK, whereas lipid‐free synthetic (s)HZ and delipidized (d)HZ did not. Consistently, both nHZ and 15‐HETE also promoted phosphorylation of MAPK‐activated protein kinase‐2, a known p38 MAPK substrate; such an effect was abolished by SB203580, a synthetic p38 MAPK inhibitor. SB203580 also abrogated nHZ‐dependent and 15‐HETE‐dependent enhancement of MMP‐9 mRNA and protein (latent and activated forms) levels in cell lysates and supernatants. Collectively, these data suggest that in human monocytes, nHZ and 15‐HETE upregulate MMP‐9 expression and secretion through activation of p38 MAPK pathway. The present work provides new evidence on mechanisms underlying MMP‐9 deregulation in malaria, which might be helpful to design new specific drugs for adjuvant therapy in complicated malaria. Copyright © 2013 John Wiley & Sons, Ltd.     

Establishment and characterization of a normal melanocyte cell line derived from pig skin

Several minipig strains develop spontaneous malignant melanoma. As a first step toward the analysis of genes involved in the tumoral progression of melanoma in these animal models, we developed culture conditions for pig melanocytes whereby melanocytes from normal epidermis can be isolated directly onto mitotically inactivated keratinocytes in Eagle's minimal essential medium supplemented with fetal calf serum, tetradecanoyl phorbol acetate (TPA) and cholera toxin. We also derived an immortal line of pigmented melanocytes from the epidermis of a healthy Meishan pig. This cell line, designated PigMel, retains differentiation function in culture, dependence on TPA and cholera toxin and a diploid chromosome number. PigMel melanocytes exhibit morphological and molecular characteristics common to normal mammalian skin melanocytes.     

Morphofunctional study of 12‐O‐tetradecanoyl‐13‐phorbol acetate (TPA)‐induced differentiation of U937 cells under exposure to a 6 mT static magnetic field

This study deals with the morphofunctional influence of 72 h exposure to a 6 mT static magnetic field (SMF) during differentiation induced by 50 ng/ml 12‐O‐tetradecanoyl‐13‐phorbol acetate (TPA) in human leukaemia U937 cells. The cell morphology of U937 cells was investigated by optic and electron microscopy. Specific antibodies and/or molecules were used to label CD11c, CD14, phosphatidylserine, F‐actin and to investigate the distribution and activity of lysosomes, mitochondria and SER. [Ca²⁺]_i was evaluated with a spectrophotometer. The degree of differentiation in SMF‐exposed cells was lower than that of non‐exposed cells, the difference being exposure time‐dependent. SMF‐exposed cells showed cell shape and F‐actin modification, inhibition of cell attachment, appearance of membrane roughness and large blebs and impaired expression of specific macrophagic markers on the cell surface. The intracellular localization of SER and lysosomes was only partially affected by exposure. A significant localization of mitochondria with an intact membrane potential at the cell periphery in non‐exposed, TPA‐stimulated cells was observed; conversely, in the presence of SMF, mitochondria were mainly localised near the nucleus. In no case did SMF exposure affect cell viability. The sharp intracellular increase of [Ca²⁺]_i could be one of the causes of the above‐described changes. Bioelectromagnetics 30:352–364, 2009. © 2009 Wiley‐Liss, Inc.     

Synthesis of New Potential Lipophilic Co‐Drugs of 2‐Chloro‐2′‐deoxyadenosine (Cladribine, 2‐CdA,Mavenclad®, Leustatin®) and 6‐Azauridine (z6U) with Valproic Acid

2‐Chloro‐2′‐deoxyadenosine (cladribine, 1 ) was acylated with valproic acid ( 2 ) under various reaction conditions yielding 2‐chloro‐2′‐deoxy‐3′,5′‐O‐divalproyladenosine ( 3 ) as well as the 3′‐O‐ and 5′‐O‐monovalproylated derivatives, 2‐chloro‐2′‐deoxy‐3′‐O‐valproyladenosine ( 4 ) and 2‐chloro‐2′‐deoxy‐5′‐O‐valproyladenosine ( 5 ), as new co‐drugs. In addition, 6‐azauridine‐2′,3′‐O‐(ethyl levulinate) ( 8 ) was valproylated at the 5′‐OH group (→ 9 ). All products were characterized by ¹H‐ and ¹³C‐NMR spectroscopy and ESI mass spectrometry. The structure of the by‐product 6 (N‐cyclohexyl‐N‐(cyclohexylcarbamoyl)‐2‐propylpentanamide), formed upon valproylation of cladribine in the presence of N,N‐dimethylaminopyridine and dicyclohexylcarbodiimide, was analyzed by X‐ray crystallography. Cladribine as well as its valproylated co‐drugs were tested upon their cancerostatic/cancerotoxic activity in human astrocytoma/oligodendroglioma GOS‐3 cells, in rat malignant neuro ectodermal BT4Ca cells, as well as in phorbol‐12‐myristate 13‐acetate (PMA)‐differentiated human THP‐1 macrophages. The most important result of these experiments is the finding that only the 3′‐O‐valproylated derivative 4 exhibits a significant antitumor activity while the 5′‐O‐ as well as the 3′,5′‐O‐divalproylated cladribine derivatives 3 and 5 proved to be inactive.     

MMP‐2 and MMP‐9 as prognostic markers for the early detection of urinary bladder cancer

The present study assessed protein and gene expression levels of tissue inhibitor of metalloproteinase‐2 (TIMP‐2), matrix metalloproteinase‐2 (MMP‐2), and MMP‐9 in urine and blood samples of 50 patients with bladder carcinoma. The expression of TIMP‐2, MMP‐2, and MMP‐9 levels with tumor stage and grade was also assessed. Results showed that the expression levels of MMP‐2 and MMP‐9 in both blood and urine were significantly elevated in group 1 when compared with groups 2 and 3 healthy subjects. The discriminatory ability in the diagnosis of bladder carcinoma of MMP‐2 and MMP‐9 expression was confirmed by receiver operating characteristic curve analysis that revealed a sensitivity and specificity of 100%. MMP‐2 and MMP‐9 levels were not correlated with grade or stage of the tumor. With respect to TIMP‐2 blood and urine levels, results showed a significant decrease in gene expression levels in bladder carcinoma group, whereas, TIMP‐2 protein showed a significant increase in bladder carcinoma.     

Distinct Melanogenic Response of Human Melanocytes in Mono‐culture,in Co‐Culture with Keratinocytes and in Reconstructed Epidermis,to UV Exposure

Striking differences are observed in the melanogenic response of normal human melanocytes to UVA and UVB irradiation depending on culture conditions and the presence of keratinocytes. Exposure of melanocytes co‐cultured with keratinocytes to UVB irradiation triggered, already at low doses (5 mJ/cm²), an increase in melanin synthesis whereas in melanocyte mono‐cultures, UVB doses up to 50 mJ/cm² had no melanogenic effect. Unlike UVB, UVA exposure caused the same melanogenic response in both mono‐ and co‐cultures. Removing certain keratinocyte growth factors from the co‐culture medium abolished the melanogenic response to UVB, but not to UVA exposure. When integrated into the basal layer of a reconstructed human epidermis, human melanocytes similarly reacted to UVA and UVB irradiation as in vivo by increasing their production and transfer of melanin to the neighboring keratinocytes which resulted in a noticeable tanning of the reconstructed epidermis. The presence of a dense stratum corneum, known to scatter and absorb UV light, is responsible for higher minimal UVB and UVA doses required to trigger a melanogenic response in the reconstructed epidermis compared to keratinocyte–melanocyte co‐cultures. Furthermore, an immediate tanning response was observed in the pigmented epidermis following UVA irradiation. From these results we conclude that: (i) keratinocytes play an important role in mediating UVB‐induced pigmentation, (ii) UVA‐induced pigmentation is the result of a rather direct effect on melanocytes and (iii) reconstructed pigmented epidermis is the most appropriate model to study UV‐induced pigmentation in vitro.     

12‐O‐tetradecanoylphorbol‐13‐acetate‐induced invasion/migration of glioblastoma cells through activating PKCα/ERK/NF‐κB‐dependent MMP‐9 expression

An increase in MMP‐9 gene expression and enzyme activity with stimulating the migration of GBM8401 glioma cells via wound healing assay by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) was detected in glioblastoma cells GBM8401. TPA‐induced translocation of protein kinase C (PKC)α from the cytosol to membranes, and migration of GBM8401 elicited by TPA was suppressed by adding the PKCα inhibitors, GF109203X and H7. Activation of extracellular signal‐regulated kinase (ERK) and c‐Jun‐N‐terminal kinase (JNK) by TPA was identified, and TPA‐induced migration and MMP‐9 activity was significantly blocked by ERK inhibitor PD98059 and U0126, but not JNK inhibitor SP600125. Activation of NF‐κB protein p65 nuclear translocation and IκBα protein phosphorylation with increased NF‐κB‐directed luciferase activity by TPA were observed, and these were blocked by the PD98059 and IkB inhibitor BAY117082 accompanied by reducing migration and MMP‐9 activity induced by TPA in GBM8401 cells. Transfection of GBM8401 cells with PKCα siRNA specifically reduced PKCα protein expression with blocking TPA‐induced MMP‐9 activation and migration. Additionally, suppression of TPA‐induced PKCα/ERK/NK‐κB activation, migration, and MMP‐9 activation by flavonoids including kaempferol (Kae; 3,5,7,4′‐tetrahydroxyflavone), luteolin (Lut; 5,7,3′4′‐tetrahydroxyflavone), and wogonin (Wog; 5,7‐dihydroxy‐8‐methoxyflavone) was demonstrated, and structure–activity relationship (SAR) studies showed that hydroxyl (OH) groups at C4′ and C8 are critical for flavonoids' action against MMP‐9 enzyme activation and migration/invasion of glioblastoma cells elicited by TPA. Application of flavonoids to prevent the migration/invasion of glioblastoma cells through blocking PKCα/ERK/NF‐κB activation is first demonstrated herein. J. Cell. Physiol. 225: 472–481, 2010. © 2010 Wiley‐Liss, Inc.