PDGF-PDGFR network differentially regulates the fate,migration, proliferation,and cell cycle progression of myogenic cells

Platelet-derived growth factors (PDGFs) regulate embryonic development, tissue regeneration, and wound healing through their binding to PDGF receptors, PDGFRα and PDGFRβ. However, the role of PDGF signaling in regulating muscle development and regeneration remains elusive, and the cellular and molecular responses of myogenic cells are understudied. Here, we explore the PDGF-PDGFR gene expression changes and their involvement in skeletal muscle myogenesis and myogenic fate. By surveying bulk RNA sequencing and single-cell profiling data of skeletal muscle stem cells, we show that myogenic progenitors and muscle stem cells differentially express PDGF ligands and PDGF receptors during myogenesis. Quiescent adult muscle stem cells and myoblasts preferentially express PDGFRβ over PDGFRα. Remarkably, cell culture- and injury-induced muscle stem cell activation altered PDGF family gene expression. In myoblasts, PDGF-AB and PDGF-BB treatments activate two pro-chemotactic and pro-mitogenic downstream transducers, RAS-ERK1/2 and PI3K-AKT. PDGFRs inhibitor AG1296 inhibited ERK1/2 and AKT activation, myoblast migration, proliferation, and cell cycle progression induced by PDGF-AB and PDGF-BB. We also found that AG1296 causes myoblast G0/G1 cell cycle arrest. Remarkably, PDGF-AA did not promote a noticeable ERK1/2 or AKT activation, myoblast migration, or expansion. Also, myogenic differentiation reduced the expression of both PDGFRα and PDGFRβ, whereas forced PDGFRα expression impaired myogenesis. Thus, our data highlight PDGF signaling pathway to stimulate satellite cell proliferation aiming to enhance skeletal muscle regeneration and provide a deeper understanding of the role of PDGF signaling in non-fibroblastic cells.     

c-Src/Jak2/PDGFR/PKCδ-Dependent MMP-9 Induction Is Required for Thrombin-Stimulated Rat Brain Astrocytes Migration

Among matrix metalloproteinases (MMPs), MMP-9 has been observed in patients with brain inflammatory diseases and may contribute to the pathology of brain diseases. Thrombin has been known as a regulator of MMP-9 expression and cells migration. However, the mechanisms underlying thrombin-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) were not completely understood. Here, we demonstrated that thrombin induced the expression of pro-form MMP-9 in RBA-1 cells and cells migration which were attenuated by pretreatment with the inhibitor of receptor tyrosine kinase (Genistein), c-Src (PP1), Jak2 (AG490), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), PKCs (Ro318220), PKCδ (Rottlerin), or NF-κB (Bay11-7082) and transfection with siRNA of c-Src, PDGFR, Akt, PKCδ, ATF2, p65, IKKα, or IKKβ. In addition, thrombin-stimulated c-Src, Jak2, or PDGFR phosphorylation was inhibited by a thrombin inhibitor (PPACK), PP1, AG490, or AG1296. Thrombin further stimulated c-Src and PDGFR complex formation in RBA-1 cells. Thrombin also stimulated Akt and PKCδ phosphorylation and PKCδ translocation which were reduced by PPACK, PP1, AG490, AG1296, or LY294002. We further observed that thrombin markedly stimulated ATF2 or IκBα phosphorylation and NF-κB p65 translocation which were inhibited by Rottlerin or LY294002. Finally, thrombin stimulated in vivo binding of p65 to the MMP-9 promoter, which was reduced by pretreatment with Rottlerin or LY294002. These results concluded that in RBA-1 cells, thrombin activated a c-Src/Jak2/PDGFR/PI3K/Akt/PKCδ pathway, which in turn triggered ATF2 and NF-κB activation and ultimately induced MMP-9 expression associated with cell migration.     

Inhibition of platelet-derived growth factor actions in the embryonic testis influences normal cord development and morphology

Platelet-derived growth factors (PDGFs) are paracrine factors with roles in mesenchymal-epithelial interactions during normal and pathologic processes. Previously, PDGF and its receptor (PDGFR) have been shown to be present in perinatal, peripubertal, and adult rat testes. The role of PDGF in embryonic testicular cord formation is not known. The hypothesis tested is that PDGFs and PDGFRs are expressed during cord formation and that inhibition of their action influences normal cord formation during embryonic testis development. Embryonic Day (E) 13 gonadal organ cultures were used. Organs were cultured for 3 days and treated daily with vehicle or a PDGFR-specific tyrosine phosphorylation inhibitor (i.e., the tyrphostin AG1295 or AG1296). Vehicle-treated testes formed normal cords, whereas tyrphostin-treated testes formed "swollen cords," a phenomenon characterized by a significant decrease in the number of cords per testis area and increased cord diameter due to fusion of cords. Expression of PDGF and PDGFR in E13, E14, E16, Postnatal Day (P) 0, and P20 testes was examined. Messenger RNAs for PDGF-A and -B and PDGF alpha- and beta-receptors were expressed in isolated testes during all developmental periods examined. Immunoreactivity for PDGF was present throughout the testicular compartment at E14, restricted primarily to testicular cords at E16, and present in cells of the testicular cords with a stronger immunoreactivity in certain interstitial cell types of P0 testis. PDGFR beta-receptor immunoreactivity was primarily localized to the mesonephros of E14 organs and the testicular interstitium of E16 and P0 testes. Tyrphostins did not affect apoptotic cell number in the testis. PDGF had no effect on cell growth in P0 testis cultures. The results show that PDGFs and PDGFRs are expressed in embryonic testis during cord formation in a tissue-specific manner. Inhibition of PDGF actions does not inhibit cord formation but does alter normal cord development and morphology. The observations provide insight into the factors involved in male sex differentiation and embryonic testis development.     

Cyclic strain induces mouse embryonic stem cell differentiation into vascular smooth muscle cells by activating PDGF receptor beta

Embryonic stem (ES) cells are exposed to fluid-mechanical forces, such as cyclic strain and shear stress, during the process of embryonic development but much remains to be elucidated concerning the role of fluid-mechanical forces in ES cell differentiation. Here, we show that cyclic strain induces vascular smooth muscle cell (VSMC) differentiation in murine ES cells. Flk-1-positive (Flk-1+) ES cells seeded on flexible silicone membranes were subjected to controlled levels of cyclic strain and examined for changes in cell proliferation and expression of various cell lineage markers. When exposed to cyclic strain (4-12% strain, 1 Hz, 24 h), the Flk-1+ ES cells significantly increased in cell number and became oriented perpendicular to the direction of strain. There were dose-dependent increases in the VSMC markers smooth muscle alpha-actin and smooth muscle-myosin heavy chain at both the protein and gene expression level in response to cyclic strain, whereas expression of the vascular endothelial cell marker Flk-1 decreased, and there were no changes in the other endothelial cell markers (Flt-1, VE-cadherin, and platelet endothelial cell adhesion molecule 1), the blood cell marker CD3, or the epithelial marker keratin. The PDGF receptor beta (PDGFR beta) kinase inhibitor AG-1296 completely blocked the cyclic strain-induced increase in cell number and VSMC marker expression. Cyclic strain immediately caused phosphorylation of PDGFR beta in a dose-dependent manner, but neutralizing antibody against PDGF-BB did not block the PDGFR beta phosphorylation. These results suggest that cyclic strain activates PDGFR beta in a ligand-independent manner and that the activation plays a critical role in VSMC differentiation from Flk-1+ ES cells.     

EV71 induces VCAM-1 expression via PDGF receptor, PI3-K/Akt, p38 MAPK, JNK and NF-kappaB in vascular smooth muscle cells

Enterovirus 71 (EV71) is a widespread virus that causes severe and fatal diseases in patients, including circulation failure. The mechanisms underlying EV71-initiated intracellular signaling pathways to influence host cell functions remain unknown. In this study, we identified a requirement for PDGFR, PI3-K/Akt, p38 MAPK, JNK, and NF-kappaB in the regulation of VCAM-1 expression by rat vascular smooth muscle cells (VSMCs) in response to viral infection. EV71 induced VCAM-1 expression in a time- and viral concentration-dependent manner. Infection of VSMCs with EV71 stimulated VCAM-1 expression and phosphorylation of PDGFR, Akt, and p38 MAPK which were attenuated by AG1296, wortmannin, and SB202190, respectively. The phosphorylation of JNK stimulated by EV71 was not detected under present conditions. In contrast, JNK inhibitor SP600125 inhibited EV71-induced VCAM-1 expression. Furthermore, VCAM-1 expression induced by EV71 was significantly attenuated by a selective NF-kappaB inhibitor (helenalin). Consistently, EV71-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha as well as VCAM-1 mRNA expression was blocked by helenalin, AG1296, SB202190, SP600125, wortmannin, and LY294002. Moreover, the involvement of p38 MAPK, PI3-K/Akt, and NF-kappaB in EV71-induced VCAM-1 expression was reveled by that transfection with dominant negative plasmids of p38 MAPK, p85, Akt, NIK, IKK-alpha, and IKK-beta attenuated these responses. These findings suggest that in VSMCs, EV71-induced VCAM-1 expression was mediated through activation of PDGFR, PI3-K/Akt, p38 MAPK, JNK, and NF-kappaB pathways.     

Sphingosine-1-phosphate induces a PDGFR-dependent cell detachment via inhibiting beta1 integrin in HEK293 cells

Several different types of interactions between sphingosine-1-phosphate (S1P) receptors and platelet-derived growth factor receptor (PDGFR) have been revealed recently. In this work, we used HEK293 cells to further investigate the potential crosstalk. Interestingly, we observed that S1P specifically induced a PDGFR-dependent cell detachment in HEK293 cells, which could be inhibited by AG1296, a specific inhibitor for PDGFR. EGFR on the other hand, did not have any effect on cell detachment. The detachment was extracellular matrix (ECM) protein specific, suggesting the involvement of specific integrin molecules. When beta(1) integrin was engaged into an active state, S1P-induced cell detachment was blocked, suggesting that S1P induced an inside-out inhibitory effect on beta(1) integrin. G(i) protein and ERK activation were required for the cell detachment induced by S1P, suggesting an endogenous receptor for S1P is likely to be involved.     

Sphingosine-1-Phosphate Mediates ICAM-1-Dependent Monocyte Adhesion through p38 MAPK and p42/p44 MAPK-Dependent Akt Activation

Up-regulation of intercellular adhesion molecule-1 (ICAM-1) is frequently implicated in lung inflammation. Sphingosine-1-phosphate (S1P) has been shown to play a key role in inflammation via adhesion molecules induction, and then causes lung injury. However, the mechanisms underlying S1P-induced ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unclear. The effect of S1P on ICAM-1 expression was determined by Western blot and real-time PCR. The involvement of signaling pathways in these responses was investigated by using the selective pharmacological inhibitors and transfection with siRNAs. S1P markedly induced ICAM-1 expression and monocyte adhesion which were attenuated by pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), PI3K (LY294002), or AP-1 (Tanshinone IIA) and transfection with siRNA of S1PR1, S1PR3, c-Src, EGFR, PDGFR, p38, p42, JNK1, c-Jun, or c-Fos. We observed that S1P-stimulated p42/p44 MAPK and p38 MAPK activation was mediated via a c-Src/EGFR and PDGFR-dependent pathway. S1P caused the c-Src/EGFR/PDGFR complex formation. On the other hand, we demonstrated that S1P induced p42/p44 MAPK and p38 MAPK-dependent Akt activation. In addition, S1P-stimulated JNK1/2 phosphorylation was attenuated by SP600125 or PP1. Finally, S1P enhanced c-Fos mRNA levels and c-Jun phosphorylation. S1P-induced c-Jun activation was reduced by PP1, AG1478, AG1296, U0126, SP600125, SB202190, or LY294002. These results demonstrated that S1P-induced ICAM-1 expression and monocyte adhesion were mediated through S1PR1/3/c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt-dependent AP-1 activation.     

PDGFRβ triggered by bFGF promotes the proliferation and migration of endothelial progenitor cells via p-ERK signalling

We have examined the effects of bFGF (basic fibroblast growth factor) on p-ERK (phosphorylated extracellular signal-regulated kinase) through PDGFRβ (platelet-derived growth factor receptor β) in the proliferation and migration of EPCs (endothelial progenitor cells). EPC migration was detected using the Transwell system. The expression of PDGFRβ mRNA and protein, total ERK and p-ERK proteins was respectively assessed by real-time PCR and Western blottings. bFGF promote the proliferation and migration of EPCs, the effects of bFGF being implemented by activating ERK signalling through the expression of PDGFRβ, whereas an anti-bFGF antibody and inhibitor of PDGF (platelet-derived growth factor) receptor kinase (AG1296) could respectively decrease the expression of PDGFRβ mRNA and protein and p-ERK protein. Total ERK protein did not change under the same experimental conditions, and an inhibitor of p-ERK (PD98059) inhibited the proliferation and migration of EPCs. The findings strongly suggest that a PDGFRβ/p-ERK signalling pathway triggered by bFGF plays an important role in the proliferation and migration of EPCs.     

Interactions between cadherin-11 and platelet-derived growth factor receptor-alpha signaling link cell adhesion and proliferation

Cadherins are homophilic cell-to-cell adhesion molecules that help cells respond to environmental changes. Newly formed cadherin junctions are associated with increased cell phosphorylation, but the pathways driving this signaling response are largely unknown. Since cadherins have no intrinsic signaling activity, this phosphorylation must occur through interactions with other signaling molecules. We previously reported that cadherin-11 engagement activates joint synovial fibroblasts, promoting inflammatory and degradative pathways important in rheumatoid arthritis (RA) pathogenesis. Our objective in this study was to discover interacting partners that mediate cadherin-11 signaling. Protein array screening showed that cadherin-11 extracellular binding domains linked to an Fc domain (cad11Fc) induced platelet-derived growth factor (PDGFR)-α phosphorylation in synovial fibroblasts and glioblastoma cells. PDGFRs are growth factor receptor tyrosine kinases that promote cell proliferation, survival, and migration in mesodermally derived cells. Increased PDGFR activity is implicated in RA pathology and associates with poor prognosis in several cancers, including sarcoma and glioblastoma. PDGFRα activation by cadherin-11 signaling promoted fibroblast proliferation, a signaling pathway independent from cadherin-11-stimulated IL-6 or matrix metalloproteinase (MMP)-3 release. PDGFRα phosphorylation mediated most of the cad11Fc-induced phosphatidyl-3-kinase (PI3K)/Akt activation, but only part of the mitogen-activated protein kinase (MAPK) response. PDGFRα-dependent signaling did not require cell cadherin-11 expression. Rather, cad11Fc immunoprecipitated PDGFRα, indicating a direct interaction between cadherin-11 and PDGFRα extracellular domains. This study is the first to report an interaction between cadherin-11 and PDGFRα and adds to our growing understanding that cadherin-growth factor receptor interactions help balance the interplay between tissue growth and adhesion.     

Metformin Protects Cardiomyocyte from Doxorubicin Induced Cytotoxicity through an AMP-Activated Protein Kinase Dependent Signaling Pathway: An In Vitro Study

Doxorubicin (Dox) is one of the most widely used antitumor drugs, but its cumulative cardiotoxicity have been major concerns in cancer therapeutic practice for decades. Recent studies established that metformin (Met), an oral anti-diabetic drug, provides protective effects in Dox-induced cardiotoxicity. Met has been shown to increase fatty acid oxidation, an effect mediated by AMP activated protein kinase (AMPK). Here we delineate the intracellular signaling factors involved in Met mediated protection against Dox-induced cardiotoxicity in the H9c2 cardiomyoblast cell line. Treatment with low dose Met (0.1 mM) increased cell viabilities and Ki-67 expressions while decreasing LDH leakages, ROS generations and [Ca²⁺]_i. The protective effect was reversed by a co-treatment with compound-C, an AMPK specific inhibitor, or by an over expression of a dominant-negative AMPKα cDNA. Inhibition of PKA with H89 or a suppression of Src kinase by a small hairpin siRNA also abrogated the protective effect of the low dose Met. Whereas, with a higher dose of Met (1.0 mM), the protective effects were abolished regardless of the enhanced AMPK, PKA/CREB1 and Src kinase activity. In high dose Met treated cells, expression of platelet-derived growth factor receptor (PDGFR) was significantly suppressed. Furthermore, the protective effect of low dose Met was totally reversed by co-treatment with AG1296, a PDGFR specific antagonist. These data provide in vitro evidence supporting a signaling cascade by which low dose Met exerts protective effects against Dox via sequential involvement of AMPK, PKA/CREB1, Src and PDGFR. Whereas high dose Met reverses the effect by suppressing PDGFR expression.     

β2-Adrenergic receptor-induced transactivation of epidermal growth factor receptor and platelet-derived growth factor receptor via Src kinase promotes rat cardiomyocyte survival

Chronic stimulation of the β-AR (adrenergic receptor) promotes apoptosis of cardiomyocytes, which is implicated in cardiac dysfunction. β1-AR and β2-AR are the main subtypes of β-AR that exert distinct effects on the survival of cardiomyocytes. To clarify the physiological roles of β1-AR and β2-AR in cardiomyocytes, the effects of β1-AR or β2-AR knockdown on the survival of H9c2 cardiomyocytes was investigated. Knockdown of β2-AR, but not β1-AR, suppressed the phosphorylation of EGFR (epidermal growth factor receptor) and PDGFR (platelet-derived growth factor receptor) induced by ISO (isoprenaline). The EGFR inhibitor, AG1478, attenuated ERK (extracellular-signal-regulated kinase) activation and partially decreased cell survival. Pretreatment with AG1296, a PDGFR inhibitor, abolished ISO-induced Akt (also known as protein kinase B) phosphorylation and led to a decrease in cell viability. In addition, the Src tyrosine kinase inhibitor, PP2, blocked ISO-mediated both Akt and ERK activation and heavily suppressed viability. Accordingly, in primary neonatal rat cardiomyocytes, the β2-AR inhibitor, but not the β1-AR inhibitor, abrogated the transactivation of EGFR and PDGFR, which was respectively related to Akt and ERK activation. The results show that β2-AR transactivates PDGFR and EGFR, thereby promoting survival of cardiomyocytes.     

Comparison of long-term retinoic acid-based neural induction methods of bone marrow human mesenchymal stem cells

The differentiation of human mesenchymal stem cells (hMSCs) into neural cells in vitro provides a potential tool to be utilized for cell therapy of neurodegenerative disorders. Although previous studies repeated different protocols for the induction of neural cells from hMSCs in vitro, the results were not in complete agreement. In this study, we have attempted to compare three of these neural induction methods; retinoic acid (RA) treatment, RA treatment in serum reduced conditions, and treatment using other chemical compounds (dimethyl sulfoxide and potassium chloride) along with RA by real-time cell analysis and immunofluorescent staining of neural markers. RA treatment led to a slow progression of cells into neural-like morphology with the expression of neural protein neurofilament whereas reducing serum during RA treatment caused a much more extended differentiation process. Additionally, neural-like morphology was persistent in the later periods of differentiation in RA treatment. On the other hand, chemical induction caused cell shrinkages mimicking neural-like morphology in a short time and loss of this morphology along with increased cell death in later periods. Among the three methods compared, RA treatment was the most reliable one in terms of stability of differentiation and neural protein expressions.     

MicroRNA-30c contributes to the development of hypoxia pulmonary hypertension by inhibiting platelet-derived growth factor receptor β expression

Pulmonary arterial hypertension (PAH) is characterized by excessive proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs). MicroRNAs have been implicated in the regulation of cell proliferation and might be implicated in the etiology of PAH. Data from in vivo and in vitro cell culture models showed that hypoxia inhibits microRNA-30c (miR-30c) expression in PASMCs. Inhibition of miR-30c by either hypoxia or AMO-30c results in PASMC proliferation (cell viability, 5-bromo-2-deoxyuridine (BrdU) incorporation, proliferating cell nuclear antigen, Ki67, and tubulin polymerization) and the inhibition of apoptosis (cell cycle progression, Cyclin A and Cyclin D, and TUNEL staining). Moreover, down-regulation of miR-30c also results in the phenotype switch from contractile to synthetic PASMC (SM22α and Calponin, osteopontin expression, and wound healing assay). In contrast, these effects were reversed by the application of an miR-30c mimetic under hypoxic conditions. Mechanically, miR-30c inhibited the platelet-derived growth factor receptor β (PDGFRβ) expression by directly binding to the 3′ untranslated region of PDGFRβ mRNA (luciferase reporter assays, and PDGFRβ-masking antisense oligodeoxynucleotides). Pharmacological inhibition of PDGFR by AG-1296 displayed similar effects to the miR-30c mimetic. These data suggest that the down-regulation of miR-30c accounts for the up-regulation of PDGFRβ expression, and subsequent activation of PDGF signaling results in the hypoxia-induced PASMC proliferation and phenotype switching. Therefore, increasing miR-30c expression levels could be explored as a potential new therapy for hypoxia-induced PAH.     

Critical roles of Src family tyrosine kinases in excitatory neuronal differentiation of cultured embryonic stem cells

Embryonic stem (ES) cells have been tested for potential cell transplantation therapy for CNS disorders. Understanding their differentiation mechanism and identifying factors involved in driving excitatory and inhibitory neuron lineages should enhance the efficacy and efficiency of the cell transplantation therapy. We tested the hypothesis that selective expression of Src family tyrosine kinases is required for phenotype-specific differentiation and functional maturation of ES cell derived neurons. Cultured mouse pluripotent ES cells were treated with retinoic acid (RA) to induce neural differentiation. After RA induction, neurons derived from ES cells showed significant neurite growth, increased expression of Src, Fyn and Lck and an extension of Src kinase expression from cell body to neurite processes. ES cell derived neuron-like cells expressed neurofilament, synaptophysin, glutamate receptors, NMDA and kainate currents, became vulnerable to excitotoxicity and formed functional excitatory synapses. These developmental events were blocked or attenuated when cells were grown in the presence of Src family kinase inhibitor PP2. However, there was no change in the expression of GABAergic-specific protein GAD67 during PP2 treatment. Our data suggest that Src tyrosine kinases are involved in the terminal differentiation of excitatory neuronal phenotype during ES cell neural differentiation after RA induction.     

Dickkopf‐3 alters the morphological response to retinoic acid during neuronal differentiation of human embryonal carcinoma cells

Dickkopf‐3 (Dkk‐3) and Dkkl‐1 (Soggy) are secreted proteins of poorly understood function that are highly expressed in subsets of neurons in the brain. To explore their potential roles during neuronal development, we examined their expression in Ntera‐2 (NT2) human embryonal carcinoma cells, which differentiate into neurons upon treatment with retinoic acid (RA). RA treatment increased the mRNA and protein levels of Dkk‐3 but not of Dkkl‐1. Ectopic expression of both Dkk‐3 and Dkkl‐1 induced apoptosis in NT2 cells. Gene silencing of Dkk‐3 did not affect NT2 cell growth or differentiation but altered their response to RA in suspension cultures. RA treatment of NT2 cells cultured in suspension resulted in morphological changes that led to cell attachment and flattening out of cell aggregates. Although there were no significant differences in the expression levels of cell adhesion molecules in control and Dkk‐3‐silenced cells, this morphological response was not observed in Dkk‐3‐silenced cells. These findings suggest that Dkk‐3 plays a role in the regulation of cell interactions during RA‐induced neuronal differentiation. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1243–1254, 2014     

Potential role of follicle-stimulating hormone (FSH) and transforming growth factor (TGFβ1) in the regulation of ovarian angiogenesis

Angiogenesis occurs during ovarian follicle development and luteinization. Pituitary secreted FSH was reported to stimulate the expression of endothelial mitogen VEGF in granulosa cells. And, intraovarian cytokine transforming growth factor (TGF)β1 is known to facilitate FSH‐induced differentiation of ovarian granulosa cells. This intrigues us to investigate the potential role of FSH and TGFβ1 regulation of granulosa cell function in relation to ovarian angiogenesis. Granulosa cells were isolated from gonadotropin‐primed immature rats and treated once with FSH and/or TGFβ1 for 48 h, and the angiogenic potential of conditioned media (granulosa cell culture conditioned media; GCCM) was determined using an in vitro assay with aortic ring embedded in collagen gel and immunoblotting. FSH and TGFβ1 increased the secreted angiogenic activity in granulosa cells (FSH + TGFβ1 > FSH ≈ TGFβ1 > control) that was partly attributed to the increased secretion of pro‐angiogenic factors VEGF and PDGF‐B. This is further supported by the evidence that pre‐treatment with inhibitor of VEGF receptor‐2 (Ki8751) or PDGF receptor (AG1296) throughout or only during the first 2‐day aortic ring culture period suppressed microvessel growth in GCCM‐treated groups, and also inhibited the FSH + TGFβ1‐GCCM‐stimulated release of matrix remodeling‐associated gelatinase activities. Interestingly, pre‐treatment of AG1296 at late stage suppressed GCCM‐induced microvessel growth and stability with demise of endothelial and mural cells. Together, we provide original findings that both FSH and TGFβ1 increased the secretion of VEGF and PDGF‐B, and that in turn up‐regulated the angiogenic activity in rat ovarian granulosa cells. This implicates that FSH and TGFβ1 play important roles in regulation of ovarian angiogenesis during follicle development. J. Cell. Physiol. 226: 1608–1619, 2011. © 2010 Wiley‐Liss, Inc.     

A splicing factor phosphorylated by protein kinase A is increased in HL60 cells treated with retinoic acid

Retinoic acid (RA) induces the differentiation of human promyelocytic leukemia HL60 cells into granulocytic cells and inhibits proliferation. Certain of actions of RA are mediated by RA nuclear receptors that regulate gene expression. However, it is also known that direct protein modification by RA (retinoylation) can occur. One such retinoylated protein in HL60 cells is a regulatory subunit of protein kinase A (PKA), which is increased in the nucleus following RA treatment and which then increases phosphorylation of other nuclear proteins. However, a complete understanding of which nuclear proteins are phosphorylated is lacking. In the current study, we employed mass spectrometry to identify one of the PKA-phosphorylated proteins as a serine/arginine-rich splicing factor 1 (SF2, SRSF1). We found that RA treatment increased the level of PKA-phosphorylated SF2 but decreased the level of SF2. While SF2 regulates myelogenous cell leukemia-1 (Mcl-1, anti-apoptotic factor), RA treatment reduced the level of Mcl-1L (full-length Mcl-1 long) and increased the level of Mcl-1S (Mcl-1 short; a short splicing variant of the Mcl-1). Furthermore, treatment with a PKA inhibitor reversed these effects on Mcl-1 and inhibited RA-induced cell differentiation. In contrast, treatment with a Mcl-1L inhibitor enhanced RA-induced cell differentiation. These results indicate that RA activates PKA in the nucleus, increases phosphorylation of SF2, raises levels of Mcl-1S and lowers levels of Mcl-1L, resulting in the induction of differentiation. RA-modified PKA may play an important role in inducing cell differentiation and suppressing cell proliferation.