RESUMO
FGF8 specifies early tooth development by directing the migration of the early tooth founder cells to the site of tooth emergence. To date the effect of the FGF8 in adult dental pulp has not been studied. We have assessed the regenerative potential of FGF8 by evaluating changes in the proteome landscape of dental pulp following short- and long-term exposure to recombinant FGF8 protein. In addition, we carried out qRT PCR analysis to determine extracellular/adhesion gene marker expression and assessed cell proliferation and mineralization in response to FGF8 treatment. 2D and mass spectrometry data showed differential expression of proteins implicated in cytoskeleton/ECM remodeling and migration, cell proliferation and odontogenic differentiation as evidenced by the upregulation of gelsolin, moesin, LMNA, WDR1, PLOD2, COPS5 and downregulation of P4HB. qRT PCR showed downregulation of proteins involved in cell-matrix adhesion such as ADAMTS8, LAMB3 and ANOS1 and increased expression of the angiogenesis marker PECAM1. We have observed that, FGF8 treatment was able to boost dental pulp cell proliferation and to enhance dental pulp mineralization. Collectively, our data suggest that, FGF8 treatment could promote endogenous healing of the dental pulp via recruitment of dental pulp progenitors as well as by promoting their angiogenic and odontogenic differentiation. SIGNIFICANCE: Dental pulp cells (DP) have been studied extensively for the purposes of mineralized tissue repair, particularly for the reconstruction of hard and soft tissue maxillofacial defects. Canonical FGF signaling has been implicated throughout multiple stages of tooth development by regulating cell proliferation, differentiation, survival as well as cellular migration. FGF8 expression is indispensible for normal tooth development and particularly for the migration of early tooth progenitors to the sites of tooth emergence. The present study provides proteome and qRT PCR data with regard to the future application and biological relevance of FGF8 in dental regenerative medicine. AUTHORS WITH ORCID: Rozaliya Tsikandelova - 0000-0003-0178-3767 Zornitsa Mihaylova - 0000-0003-1748-4489 Sébastien Planchon - 0000-0002-0455-0574 Nikolay Ishkitiev - 0000-0002-4351-5579.
Assuntos
Polpa Dentária/citologia , Fator 8 de Crescimento de Fibroblasto/farmacologia , Proteoma/metabolismo , Regeneração/efeitos dos fármacos , Adulto , Diferenciação Celular , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/fisiologia , Regulação da Expressão Gênica , Humanos , Minerais/metabolismo , Reação em Cadeia da Polimerase , Proteoma/efeitos dos fármacos , Proteoma/fisiologiaRESUMO
The Mst1 kinase is an important regulator of murine T cell adhesion, migration, proliferation, and apoptosis. In this study, we analyze mice lacking both Mst1 and Mst2 in hematopoietic cells. Compared with wild-type mice, these double knockout (DKO) mice exhibit a severe reduction in the number of mature T cells in the circulation and in secondary lymphoid organs (SLOs). CD4(+)CD8(-) and CD4(-)CD8(+) single-positive (SP) thymocytes in DKO mice resemble mature T cells of wild-type mice but undergo excessive apoptosis, and their egress from the thymus is reduced by >90%. Even when placed directly in the circulation, DKO SP thymocytes failed to enter SLOs. In SP thymocytes, deficiency of Mst1 and Mst2 abolished sphingosine-1 phosphate- and CCL21-induced Mob1 phosphorylation, Rac1 and RhoA GTP charging, and subsequent cell migration. When phosphorylated by Mst1 or Mst2, Mob1 binds and activates the Rac1 guanyl nucleotide exchanger Dock8, which is abundant in the thymus. Thus, the Mst1 and Mst2 kinases control Rho GTPase activation and the migratory responses of SP thymocytes.
Assuntos
Fator de Crescimento de Hepatócito/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Timócitos/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/química , Proteínas Adaptadoras de Transdução de Sinal , Animais , Diferenciação Celular , Movimento Celular , Quimiotaxia , Ativação Enzimática , Camundongos , Camundongos Knockout , Proteínas Quinases/metabolismo , Serina-Treonina Quinase 3RESUMO
The Rassf1-6 polypeptides each contain a Ras/Rap association domain, which enables binding to several GTP-charged Ras-like GTPases, at least in vitro or when overexpressed. The Ras/Rap association domains are followed by SARAH domains, which mediate Rassf heterodimerization with the Mst1/2 protein kinases. Rassf1A is unequivocally a tumor suppressor, and all Rassf proteins behave like tumor suppressors, exhibiting epigenetic silencing of expression in many human cancers and pro-apoptotic and/or anti-proliferative effects when re-expressed in tumor cell lines. Herein, we review the binding of the Rassf polypeptides to Ras-like GTPases and the Mst1/2 kinases and their role in Rassf function.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Dimerização , Epigênese Genética , GTP Fosfo-Hidrolases/metabolismo , Inativação Gênica , Humanos , Modelos Biológicos , Família Multigênica , Peptídeos/química , Ligação Proteica , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: MST1 and MST2 are the mammalian Ste20-related protein kinases most closely related to Drosophila Hippo, a major regulator of cell proliferation and survival during development. Overexpression of MST1 or MST2 in mammalian cells is proapototic; however, little is known concerning the physiologic regulation of the endogenous MST1/MST2 kinases, their role in mammalian cell proliferation, or the identity of the MST1/MST2 substrates critical to proliferative regulation. RESULTS: We show that MST1 and MST2 activity increases during mitosis, especially in nocodazole-arrested mitotic cells, where these kinases exhibit both an increase in both abundance and activation. MST1 and MST2 also can be activated nonphysiologically by okadaic acid or H2O2. The MOBKL1A and MOBKL1B polypeptides, homologs of the Drosophila MATS polypeptide, are identified as preferred MST1/MST2 substrates in vitro and are phosphorylated in cells in an MST1/MST2-dependent manner in mitosis and in response to okadaic acid or H2O2. MST1/MST2-catalyzed MOBKL1A/MOBKL1B phosphorylation alters the ability of MOBKL1A/MOBKL1B to bind and regulate downstream targets such as the NDR-family protein kinases. Thus, MOBKL1A/MOBKL1B phosphorylation in cells promotes MOBKL1A/MOBKL1B binding to the LATS1 kinase and enables H2O2-stimulated LATS1 activation loop phosphorylation. Most importantly, replacement of endogenous MOBKL1A/MOBKL1B by a nonphosphorylatable mutant is sufficient to accelerate cell proliferation substantially by speeding progression through G1/S as well as mitotic exit. CONCLUSIONS: These results establish that MST1 and MST2 are activated in mitosis and catalyze the mitotic phosphorylation of MOBKL1A/MOBKL1B. MOBKL1A/MOBKL1B phosphorylation, in turn, is sufficient to inhibit proliferation through actions at several points in the cell cycle.
Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Mitose/fisiologia , Fosfoproteínas/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Peróxido de Hidrogênio , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Ácido Okadáico , Fosforilação , Serina-Treonina Quinase 3RESUMO
The six human Nore1/RASSF genes encode a family of putative tumor suppressor proteins, each expressed as multiple mRNA splice variants. The predominant isoforms of these noncatalytic polypeptides are characterized by the presence in their carboxyterminal segments of a Ras-Association (RA) domain followed by a SARAH domain. The expression of the RASSF1A and Nore1A isoforms is extinguished selectively by gene loss and/or epigenetic mechanisms in a considerable fraction of epithelial cancers and cell lines derived therefrom, and reexpression usually suppresses the proliferation and tumorigenicity of these cells. RASSF1A/Nore1A can cause cell cycle delay in G1 and/or M and may promote apoptosis. The founding member, Nore1A, binds preferentially through its RA domain to the GTP-charged forms of Ras, Rap-1, and several other Ras subfamily GTPases with high affinity. By contrast, RASSF1, despite an RA domain 50% identical to Nore1, exhibits relatively low affinity for Ras-like GTPases but may associate with Ras-GTP indirectly. Each of the RASSF polypeptides, including the C. elegans ortholog encoded by T24F1.3, binds to the Ste20-related protein kinases MST1 and MST2 through the SARAH domains of each partner. The recombinant MST1/2 kinases, spontaneous dimers, autoactivate in vitro through an intradimer transphosphorylation of the activation loop, and the Nore1/RASSF1 polypeptides inhibit this process. Recombinant MST1 is strongly activated in vivo by recruitment to the membrane; the recombinant MST1 that is bound to RasG12V through Nore1A is activated; however, the bulk of MST1 is not. Endogenous complexes of MST1 with both Nore1A and RASSF1A are detectable, and Nore1A/MST1 can associate with endogenous Ras in response to serum addition. Nevertheless, the physiological functions of the Nore1/RASSF polypeptides in mammalian cells, as well as the role of the MST1/2 kinases in their growth-suppressive actions, remain to be established. The Drosophila MST1/2 ortholog hippo is a negative regulator of cell cycle progression and is necessary for developmental apoptosis. Overexpression of mammalian MST1 or MST2 promotes apoptosis, as does overexpression of mutant active Ki-Ras. Interference with the ability of endogenous MST1/2 to associate with the Nore1/RASSF polypeptides inhibits Ras-induced apoptosis. At present, however, the relevance of Ki-Ras-induced apoptosis to the physiological functions of c-Ras and to the growth-regulating actions of spontaneously occurring oncogenic Ras mutants is not known.
Assuntos
Proteínas Monoméricas de Ligação ao GTP/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma de Células Pequenas/metabolismo , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática , Fase G1/efeitos dos fármacos , Quinases do Centro Germinativo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células KB , Neoplasias/fisiopatologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Serina-Treonina Quinase 3 , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas ras/fisiologiaRESUMO
Activation of opioid receptors have been implicated in the modulation of cell proliferation and the E2F family of transcription factors may play a role in opioid inhibition of DNA synthesis. Gel shift assays and Western blotting of nuclear extracts from NG108-15 cells revealed increased E2F1 DNA binding activity and higher levels of E2F1 following activation of delta-opioid receptors. It is suggested that DADLE-induced regulation of E2F DNA binding activity involves ERKs.
Assuntos
Analgésicos Opioides/farmacologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Leucina Encefalina-2-Alanina/farmacologia , Receptores Opioides delta/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Animais , Western Blotting/métodos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Citometria de Fluxo/métodos , Imidazóis/farmacologia , Camundongos , Neuroblastoma/patologia , Ligação Proteica/efeitos dos fármacos , Piridinas/farmacologia , Timidina/farmacocinética , Trítio/farmacocinéticaRESUMO
Mouse plasmacytomas are appropriate models to study the biology of human multiple myeloma (MM). Growth of murine interleukin-6 (IL-6)-dependent hybridoma/plasmacytoma lines can be stimulated by bacterial lipopolysaccharides (LPS). However, the molecular mechanisms of this phenomenon are still not elucidated. In this study the in vitro action of bacterial LPS on the mouse IL-6-dependent B9 hybridoma/plasmacytoma cell line and two IL-6-dependent hybridomas was investigated. The involvement of different signal transduction pathways was established using specific kinase inhibitors in proliferation assays and immunoblotting analysis of the kinase activity. Selective mitogen-activated protein kinase (MAPK) kinase inhibitor PD989059 inhibited both IL-6- and LPS-induced B9 cell proliferation. In contrast, in H187 and H188 cells, PD98059 inhibited only LPS-, but not IL-6-stimulated cell growth. The kinetics of MAPK activation in all cell lines showed that phosphorylation of p42 MAPK (encoded as ERK2) but not of p44 MAPK (ERK1), was considerably increased after treatment with LPS. We found that in H187 and H188 hybridomas IL-6 induced proliferation by a different STAT3-dependent mechanism. This study demonstrates the key role of the MAPK pathway in LPS-stimulated growth of mouse IL-6-dependent plasmacytoma cells. These findings suggest the presence of signaling mechanism in MM cells inducible by bacterial mitogens and possibly mediated by Toll-like receptors (TLR)--evolutionarily conserved molecules playing a central role in the microbial recognition and initiation of the cellular innate immune response.
Assuntos
Interleucina-6/metabolismo , Lipopolissacarídeos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Plasmocitoma/metabolismo , Animais , Divisão Celular/fisiologia , Humanos , Camundongos , Salmonella/metabolismoRESUMO
MST1 (mammalian Sterile20-like 1) and MST2 are closely related Class II GC (protein Ser/Thr) kinases that initiate apoptosis when transiently overexpressed in mammalian cells. In the present study, we show that recombinant MST1/2 undergo a robust autoactivation in vitro, mediated by an intramolecular autophosphorylation of a single site [MST1(Thr183)/MST2(Thr180)] on the activation loop of an MST dimer. Endogenous full-length MST1 is activated by a variety of stressful stimuli, accompanied by the secondary appearance of a 36 kDa Thr183-phosphorylated, caspase-cleaved catalytic fragment. Recombinant MST1 exhibits only 2-5% activation during transient expression; endogenous MST1 in the cycling HeLa or KB cells has a similar low fractional activation, but 2 h incubation with okadaic acid (1 mM) results in 100% activation. Endogenous MST1 immunoprecipitated from KB cells is specifically associated with substoichiometric amounts of the growth inhibitory polypeptides RASSF1A and NORE1A (novel Ras effector 1A; a Ras-GTP-binding protein). Co-expression of RASSF1A, RASSF1C, NORE1A and NORE1B with MST1 markedly suppresses MST1(Thr183) phosphorylation in vivo and abolishes the ability of MST1 to undergo Mg-ATP-mediated autoactivation in vitro; direct addition of purified NORE1A in vitro also inhibits MST1 activation. In contrast, co-transfection of MST1 with NORE1A modified by the addition of a C-terminal CAAX motif results in a substantial increase in MST1(Thr183) phosphorylation, as does fusion of a myristoylation motif directly on to the MST1 N-terminus. Moreover, MST1 polypeptides, bound via wild-type NORE1A to Ras(G12V) (where G12V stands for Gly12Val), exhibit higher Thr183 phosphorylation compared with MST1 bound to NORE1A alone. Nevertheless, serum stimulation of KB cells does not detectably increase the activation state of endogenous MST1 or MST2 despite promoting the recruitment of the endogenous NORE1-MST1 complex to endogenous Ras. We propose that the NORE1/RASSF1 polypeptides, in addition to their role in maintaining the low activity of MST1 in vivo, direct MST1 to sites of activation and perhaps co-localization with endogenous substrates.
Assuntos
Inibidores do Crescimento/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Reguladoras de Apoptose , Células COS/enzimologia , Linhagem Celular , Chlorocebus aethiops , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Rim/citologia , Rim/embriologia , Rim/enzimologia , Fosforilação , Serina-Treonina Quinase 3RESUMO
Interleukin-6 (IL-6) mediates growth of murine B9 hybridoma/plasmacytoma cells via Ras-dependent mitogen-activated protein kinase (MAPK) pathway. Preincubation of cells with selective protein kinase C (PKC) inhibitors Gö6976 and Gö6983 leads to enhancement of IL-6-induced p44/p42 MAPK activity. The basal p44/p42 MAPK activity is also stimulated in the presence of both inhibitors. On the other hand, Gö6976 completely blocks proliferation, but Gö6983 which does not inhibit PKC(mu) has no effect on the cell growth. These findings suggest that PKC(mu) is required for proliferation and other PKC isoenzymes are involved in regulation of IL-6-dependent growth of B9 cells by negative regulation of the MAPK pathway.