RESUMO
Cardiomyocyte development switches from hyperplasmic to hypertrophic growth between postnatal days 3 and 4 in rats. The mechanisms responsible for this transition have been controversial. beta-Adrenergic receptor (betaAR) activation of mitogenic responses in vitro has been reported. We hypothesized that tonic activation of the betaAR signaling regulates cell division in neonatal cardiomyocytes via effects on signaling kinases known to be important in cell cycle regulation. The purpose of the current study was to elucidate the roles of betaAR in rat cardiomyocyte growth in vivo. We demonstrated that betaAR blockade induced a significant reduction in cardiomyocyte proliferation as measured by the BrdU labeling index. Blockade of betaAR did not affect p38 or p44/42 MAPK activities. We further demonstrated that betaAR blockade induced a prompt deactivation of the p70 ribosomal protein S6 kinase (p70 S6K). To confirm these results, we measured p70 S6K activity directly. Basal activity of p70 S6K in neonatal cardiomyocytes was fourfold higher than that of insulin-treated adult rat liver. The activity of p70 S6K was reduced by 60% within 1 min after betaAR blockade. We conclude that the betaAR are involved in regulation of neonatal cardiomyocyte proliferation and that this mitogenic control may be mediated via the p70 S6K pathway.
Assuntos
Miocárdio/citologia , Receptores Adrenérgicos beta/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Animais , Animais Recém-Nascidos , Divisão Celular , Feminino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Propranolol/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos beta/fisiologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The phosphorylation of ribosomal protein S6 is thought to be required for biosynthesis of the cell's translational apparatus, a critical component of cell growth and proliferation. We have studied the signal transduction pathways involved in hepatic S6 phosphorylation during late gestation in the rat. This is a period during which hepatocytes show a high rate of proliferation that is, at least in part, independent of mitogenic signaling pathways that are operative in mature hepatocytes. Our initial studies demonstrated that there was low basal activity of two S6 kinases in liver, S6K1 and S6K2, on embryonic day 19 (2 days preterm). In addition, insulin- and growth factor-mediated S6K1 and S6K2 activation was markedly attenuated compared with that in adult liver. Nonetheless, two-dimensional gel electrophoresis demonstrated that fetal liver S6 itself was highly phosphorylated. To characterize the fetal hepatocyte pathway for S6 phosphorylation, we went on to study the sensitivity of hepatocyte proliferation to the S6 kinase inhibitor rapamycin. Unexpectedly, administration of rapamycin to embryonic day 19 fetuses in situ did not affect hepatocyte DNA synthesis. This resistance to the growth inhibitory effect of rapamycin occurred even though S6K1 and S6K2 were inhibited. Furthermore, fetal hepatocyte proliferation was sustained even though rapamycin administration resulted in the dephosphorylation of ribosomal protein S6. In contrast, rapamycin blocked hepatic DNA synthesis in adult rats following partial hepatectomy coincident with S6 dephosphorylation. We conclude that hepatocyte proliferation in the late gestation fetus is supported by a rapamycin-resistant mechanism that can function independently of ribosomal protein S6 phosphorylation.
Assuntos
Fígado/embriologia , Fosforilação , Proteínas Ribossômicas/metabolismo , Animais , Western Blotting , Divisão Celular , Relação Dose-Resposta a Droga , Eletroforese em Gel Bidimensional/métodos , Inibidores Enzimáticos/farmacologia , Hepatócitos/metabolismo , Humanos , Fígado/enzimologia , Fígado/metabolismo , Sistema de Sinalização das MAP Quinases , Ratos , Ratos Sprague-Dawley , Proteína S6 Ribossômica , Proteínas Quinases S6 Ribossômicas/metabolismo , Sirolimo/farmacologia , Fatores de TempoRESUMO
Insulin-like growth factor-I (IGF-I) stimulates proliferation and differentiation of many cell types, including preadipocytes. We have previously shown that IGF-I stimulates proliferation of 3T3-L1 preadipocytes through activation of the extracellular regulated kinase (ERK)-1 and -2 mitogen-activated protein kinase (MAPK) pathway, and that IGF-I-stimulated MAPK is predominantly downstream of Shc, not IRS-1 phosphorylation. The Src family of nonreceptor tyrosine kinases has been shown to mediate the mitogenic effects of other growth factors that also activate Shc and the ERK-1 and -2 MAPKs. Although Src family kinases (SFK) have been implicated in IGF-I action, no specific role for SFKs in IGF-I regulation of mitogenesis has been previously demonstrated. We studied the role of SFKs in IGF-I mitogenic signaling in 3T3-L1 preadipocytes. The SFK-selective inhibitor PP1 completely inhibited both IGF-I-stimulated DNA synthesis and MAPK activation in proliferating 3T3-L1 cells. PP1 inhibited IGF-I phosphorylation of Shc but not of IRS-1. In addition, IGF-I activation of MAPK was inhibited in proliferating cells transiently transfected with a dominant-negative c-Src. Finally, the kinetics of SFK and MAPK activation by IGF-I suggest that SFKs may act upstream of MAPK. IGF-I activation of SFK members c-Src and Fyn occurred within 1 min of treatment, and activity was back to baseline by 10 min. Our previous studies found that IGF-I activation of MAPK peaked at 5 min and was also back to baseline by 10 min. Our results are the first to demonstrate that SFKs mediate IGF-I mitogenic signaling in 3T3-L1 cells and add to the growing body of evidence that SFKs play a crucial role in IGF-I action.
Assuntos
Divisão Celular/fisiologia , Fator de Crescimento Insulin-Like I/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Quinases da Família src/metabolismo , Células 3T3 , Animais , DNA/biossíntese , Inibidores Enzimáticos/farmacologia , Immunoblotting , Proteínas Substratos do Receptor de Insulina , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfoproteínas/metabolismo , Testes de Precipitina , Pirazóis/farmacologia , Pirimidinas/farmacologia , Fatores de Virulência de Bordetella/farmacologia , Quinases da Família src/antagonistas & inibidoresRESUMO
Inducible nitric oxide synthase (iNOS) may be a key mediator of intestinal injury, which varies with developmental age. One member of the mitogen-activated protein kinase (MAPK) family, p38, is involved in the lipopolysaccharide (LPS)-mediated iNOS induction. The involvement of p38 MAPK in basal and LPS-induced iNOS expression was examined in the rat intestine at two developmental ages. Neonatal (4 days postnatal) and adolescent (15 days postnatal) rats were injected with LPS (5 microg/g ip), a selective p38 inhibitor (SB 203580), or both. Tissue was removed after 4 h and 6 h for mRNA and protein analysis. iNOS mRNA and protein were markedly upregulated in the adolescent female following LPS exposure, whereas males had an attenuated response. Neonates had a minimal response. SB 203580 suppressed LPS-induced iNOS mRNA and protein in the ileum, more so in females than in males. Adolescent ileal p38 activation was constitutively high and nonresponsive to LPS. Basal and post-LPS p38 phosphorylation was low in neonatal ileum. We conclude that ileal iNOS expression is developmentally regulated and influenced by gender and that p38 is permissive for LPS effect. The developmental regulation of p38 may contribute to age-dependent variations of intestinal injury.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Íleo/crescimento & desenvolvimento , Íleo/metabolismo , Óxido Nítrico Sintase/biossíntese , Animais , Animais Recém-Nascidos , Inibidores Enzimáticos/farmacologia , Feminino , Imidazóis/farmacologia , Lipopolissacarídeos/farmacologia , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Masculino , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Piridinas/farmacologia , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Fatores Sexuais , Regulação para Cima , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
A 7.5-yr-old boy with Graves' disease, difficult to control with antithyroid medication and radioactive iodine (RAI) therapy, developed thyroid storm encephalopathy on day 13 after withdrawal of methimazole therapy, 4 days after iodine-131 treatment. We attributed his thyroid storm to withdrawal of antithyroid medication as opposed to RAI therapy. We interpret this case as indicating that there may be a need to reevaluate the duration of antithyroid medication withdrawal before RAI therapy for hyperthyroid children at increased risk for thyroid storm.
Assuntos
Antitireóideos/efeitos adversos , Doença de Graves/terapia , Radioisótopos do Iodo/efeitos adversos , Síndrome de Abstinência a Substâncias/etiologia , Crise Tireóidea/etiologia , Criança , Humanos , MasculinoRESUMO
A 7-yr-old girl presented with isosexual precocious puberty secondary to a feminizing adrenal adenoma. The adrenal tumor was found to express aromatase messenger ribonucleic acid. Enzyme kinetic studies revealed a high level of aromatase activity in the adrenal tumor, with a K(m) of 45 nmol/L and a maximum velocity of 25.6 pmol/mg.h. Aromatase activity was approximately 500-fold higher in the tumor than in adjacent normal adrenal tissue. Although histopathological examination of the tumor was most consistent with a benign adenoma, the aromatase transcripts present in the tumor corresponded to those previously associated with malignant as well as benign tumors. We consider the pattern of aromatase expression sufficient to warrant continued follow-up for tumor recurrence. Our case demonstrates that isosexual precocious puberty secondary to a feminizing adrenal tumor can be due to estrogen synthesis from the tumor itself rather than peripheral aromatization as had been previously theorized.
Assuntos
Adenoma/enzimologia , Neoplasias das Glândulas Suprarrenais/enzimologia , Aromatase/genética , Aromatase/metabolismo , Puberdade Precoce/etiologia , Adenoma/complicações , Adenoma/genética , Adenoma/cirurgia , Corticosteroides/urina , Neoplasias das Glândulas Suprarrenais/complicações , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/cirurgia , Adrenalectomia , Androgênios/sangue , Androgênios/urina , Criança , Estrogênios/sangue , Estrogênios/urina , Éxons , Feminino , Seguimentos , Humanos , Hidrocortisona/urina , Cinética , Puberdade Precoce/enzimologia , Puberdade Precoce/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estados Unidos , População BrancaRESUMO
We have shown previously that hepatocyte proliferation in the late gestation fetal rat is mediated by growth factor-independent mechanisms that are distinct from the signaling pathways that promote proliferation of adult rat hepatocytes. In the present studies, we identified six candidate growth-regulating genes that are overexpressed in fetal rat liver (embryonic day 19, 2 days pre-term) relative to adult rat liver using suppressive subtractive hybridization. These included the following: Grb10, a growth factor receptor binding protein; eps15, a growth factor receptor substrate; nuc2+, a retinoblastoma protein binding protein; cdc25B, a cell cycle tyrosine phosphatase; the peroxisome proliferator-activated receptor PPAR alpha; and a deoxyuridine triphosphatase that functions as a PPAR alpha binding partner. In every case, the ontogeny of the expression of these genes declined postnatally in a manner consistent with the transition from a fetal to an adult hepatocyte phenotype. None were found to be cell cycle-dependent, in that they did not show expression that followed perinatal changes in hepatocyte cell cycle activity. Based on our identification of these genes and previous work characterizing their role in growth regulation, we conclude that they may contribute to the mitogenic signaling phenotype of fetal rat hepatocytes.
Assuntos
Genes Reguladores , Substâncias de Crescimento/genética , Fígado/embriologia , Animais , Animais Recém-Nascidos , DNA Complementar/biossíntese , Expressão Gênica , Idade Gestacional , Fígado/fisiologia , Dados de Sequência Molecular , Fenótipo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The role of GH in the developing fetus is poorly understood. Several studies have demonstrated a limited role for GH in late fetal life. In fact, few data are available regarding GH signal transduction in the late gestation fetus. We therefore focused on a comparison of hepatic GH signaling in near-term fetal rats [embryonic day 19 (E19)] and adult rats using a combination of in vitro studies employing hepatocytes in primary culture and in vivo studies. We found that GH receptor (GHr) binding was comparable in fetal liver and adult liver. The long isoform of the GHr underwent tyrosine phosphorylation in response to GH stimulation of E19 fetal hepatocytes in a manner similar to that seen in cultured adult hepatocytes. Furthermore, downstream signaling via the Janus kinase-2 tyrosine kinase, STAT1 (signal transducer and activator of transcription), and STAT5 was also intact in both, as demonstrated by the tyrosine phosphorylation of these signaling proteins. To confirm the relevance of these findings to the in vivo situation, GH was directly administered by ip injection to E 19 fetal and adult rats. In both cases, tyrosine phosphorylation of STAT5 was markedly and rapidly induced. Finally, transfection of E19 fetal hepatocytes with GH-responsive reporter elements [Spi2.1(-275/+85)-CAT and 8xGHRE-TKCAT] demonstrated intact transcriptional regulation. Our data indicate that GHr abundance and activity as well as downstream GH signaling are similar in the late gestation fetal rat and in the adult and that these mechanisms appear capable of supporting physiological GH functions in the developing liver.
Assuntos
Feto/fisiologia , Hormônio do Crescimento/fisiologia , Fígado/embriologia , Transdução de Sinais/fisiologia , Envelhecimento/fisiologia , Animais , Células Cultivadas , Proteínas de Ligação a DNA/fisiologia , Feto/metabolismo , Idade Gestacional , Hormônio do Crescimento Humano/farmacologia , Humanos , Injeções Intraperitoneais , Fígado/citologia , Fígado/fisiologia , Fosforilação , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores da Somatotropina/metabolismo , Fator de Transcrição STAT1 , Transativadores/fisiologia , Transcrição Gênica , Tirosina/metabolismoRESUMO
Hepatocytes undergo marked changes in proliferation during normal liver development. In order to elucidate the mechanism for these changes, we examined the ontogeny of expression for the known cyclin-dependent kinase inhibitors (CKIs), p15(Ink4b), p16(Ink4a), p18(Ink4c), p19(Ink4d), p21(Cip1), p27(Kip1) and p57(Kip2). All except p16(Ink4a) were expressed at some time between late gestation and adulthood. The mRNA and protein expression patterns for p15(Ink4b) and p57(Kip2) were consistent with a role for these CKIs in the regulation of hepatocyte proliferation. Specifically, p57(Kip2) may contribute to hepatocyte growth arrest that occurs in term fetuses, while p15(Ink4b) may contribute to the maintenance of adult hepatocytes in a quiescent state. These results assign a possible role to two CKIs not previously identified as involved in hepatocyte cell cycle control.
Assuntos
Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Fígado/metabolismo , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor , Animais , Western Blotting , Proteínas de Transporte/análise , Proteínas de Transporte/fisiologia , Inibidor de Quinase Dependente de Ciclina p15 , Inibidor p16 de Quinase Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p18 , Inibidor de Quinase Dependente de Ciclina p19 , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Inibidor de Quinase Dependente de Ciclina p57 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/genética , Inibidores Enzimáticos/análise , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hepatócitos/química , Hepatócitos/citologia , Hepatócitos/metabolismo , Imuno-Histoquímica , Fígado/química , Fígado/embriologia , Masculino , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/análise , Proteínas Nucleares/fisiologia , RNA/genética , RNA/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
During normal development in the rat, hepatocytes undergo marked changes in the rate of proliferation. We have previously observed transient G(1) growth arrest at term, re-activation of proliferation immediately after birth, and a gradual transition to the quiescent adult hepatocyte phenotype after postnatal day 4. We hypothesized that these changes in proliferation are due in part to growth inhibitory effects mediated by the p38 mitogen-activated protein kinase pathway. p38 kinase activity measurements showed an inverse relationship with hepatocyte proliferation during the perinatal and postnatal transitions, whereas p38 content remained constant. Anisomycin activated the p38 pathway in fetal hepatocyte cultures while inducing growth inhibition that was sensitive to the p38 inhibitor, SB203580. Activation of p38 in these cultures, via transient transfection with a constitutively active form of its upstream kinase MKK6, also inhibited DNA synthesis as well as reducing cyclin D1 content. Transfection with inactive MKK6 did neither. Furthermore, MKK6-induced growth arrest was sensitive to SB203580. Finally, administration of SB203580 to near-term fetal rats in utero abrogated the transient hepatocyte growth arrest that occurs at term. These findings indicate a role for the p38 mitogen-activated protein kinase pathway in the physiological regulation of hepatocyte proliferation during normal development in the rat.
Assuntos
Hepatócitos/citologia , Fígado/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Envelhecimento , Animais , Anisomicina/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Divisão Celular , Células Cultivadas , Desenvolvimento Embrionário e Fetal , Inibidores Enzimáticos/farmacologia , Feto , Idade Gestacional , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Imidazóis/farmacologia , Cinética , Fígado/embriologia , Fígado/crescimento & desenvolvimento , MAP Quinase Quinase 6 , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Transfecção , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Hepatocytes are capable of marked changes in proliferation in response to various physiological and pathophysiological stimuli. Although the changes in adult hepatocyte growth regulation that accompany reduction of liver mass, liver injury, and liver carcinogenesis have come under intense scrutiny, the regulation of hepatocyte growth during the latter stages of development is largely uncharacterized. We have examined hepatic cell cycle control in the developing rat. Analysis of term (fetal day 21) liver and cultured, term hepatocytes revealed G0-G1 growth-arrested cells relative to preterm (fetal day 19) liver and isolated hepatocytes. G1 cyclin-dependent kinase (CDK) activity was correlated with growth arrest at term in both in vivo and in vitro studies. The decline in CDK activity at term could not be attributed to a change in CDK protein content. Rather, the decline in CDK activity was associated with a concomitant decline in cyclin D1 protein content. However, cyclin D1 mRNA levels did not correlate with protein levels. Cyclin D1 mRNA was present at a higher level in adult livers, in which cyclin D1 protein was absent, than in fetal livers. We also examined the phosphorylation (activation) state of p38 mitogen-activated protein kinase, a potential hepatocyte-growth regulator and modulator of cyclin D1 content. p38 activity was inversely related to cyclin D1 content during liver development and regeneration. These data indicate that a posttranscriptional mechanism regulating cyclin D1 content is involved in the temporary hepatocyte growth arrest seen in the perinatal period and in the maintenance of adult hepatocytes in a quiescent state. We speculate that this posttranscriptional regulation may be downstream from the p38 mitogen-activated protein kinase pathway.
Assuntos
Ciclo Celular , Ciclina D1/fisiologia , Fígado/embriologia , Processamento Pós-Transcricional do RNA , Fatores Etários , Animais , Northern Blotting , Western Blotting , Células Cultivadas , Ciclina D1/genética , Ciclina D1/metabolismo , Ciclina E/metabolismo , Ciclina E/fisiologia , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Fígado/crescimento & desenvolvimento , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Testes de Precipitina , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Regeneração , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Insulin-like growth factor-I (IGF-I) stimulates mitogenesis in proliferating preadipocytes, but when cells reach confluence and become growth arrested, IGF-I stimulates differentiation into adipocytes. IGF-I induces signaling pathways that involve IGF-I receptor-mediated tyrosine phosphorylation of Shc and insulin receptor substrate 1 (IRS-1). Either of these adaptor proteins can lead to activation of the three-kinase cascade ending in activation of the extracellular signal-regulated kinase 1 and -2 (ERK-1 and -2) mitogen-activated protein kinases (MAPKs). Several lines of evidence suggest that activation of MAPK inhibits 3T3-L1 preadipocyte differentiation. We have shown that IGF-I stimulation of MAPK activity is lost as 3T3-L1 preadipocytes begin to differentiate. This change in MAPK signaling coincides with loss of IGF-I-mediated Shc, but not IRS-1, tyrosine phosphorylation. We hypothesized that down-regulation of MAPK via loss of proximal signaling through Shc is an early component in the IGF-I switch from mitogenesis to differentiation in 3T3-L1 preadipocytes. Treatment of subconfluent cells with the MEK inhibitor PD098059 inhibited both IGF-I-activation of MAPK as well as 3H-thymidine incorporation. PD098059, in the presence of differentiation-inducing media, accelerated differentiation in subconfluent cells as measured by expression of adipocyte protein-2 (aP-2), peroxisome proliferator-activated receptor gamma (PPARgamma) and lipoprotein lipase (LPL). Transient transfection of subconfluent cells with Shc-Y317F, a dominant-negative mutant, attenuated IGF-I-mediated MAPK activation, inhibited DNA synthesis, and accelerated expression of differentiation markers aP-2, PPARgamma, and LPL. We conclude that signaling through Shc to MAPK plays a critical role in mediating IGF-I-stimulated 3T3-L1 mitogenesis. Our results suggest that loss of the ability of IGF-I to activate Shc signaling to MAPK may be an early component of adipogenesis in 3T3-L1 cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Adipócitos/citologia , Diferenciação Celular , Fator de Crescimento Insulin-Like I/farmacologia , Mitose , Proteínas/fisiologia , Células-Tronco/citologia , Células 3T3 , Animais , Northern Blotting , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Meios de Cultura , DNA/biossíntese , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Mutação , Proteínas/genética , Proteínas Recombinantes de Fusão/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , TransfecçãoRESUMO
Mitogenic signaling involves protein kinases that phosphorylate the mitogen-activated protein kinase (MAPK) activator, MEK. In rats, basal hepatic MEK kinase activity is low in vivo in both adult rats and late gestation fetal rats, and is markedly stimulated by intraperitoneal administration of epidermal growth factor (EGF). The level of stimulated MEK phosphorylating activity is approximately 15 times higher in fetal liver than in adult liver. To identify regulated forms of the two categories of MEK kinase, Raf and MEKK, Western immunoblotting, immunoprecipitation kinase assays and immunodepletion studies were performed. Western immunoblotting confirmed that Raf-1, A-Raf, B-Raf, MEKK1 and MEKK2 were present at similar levels in E19 and adult liver. However, specific immunoprecipitation kinase assays did not detect any kinases that could account for marked EGF sensitivity or the higher level of activity in E19 fetuses. Immunodepletion studies produced a marked reduction in immunoreactive Raf/MEKK content and activity, but a minimal decrease in the ability of chromatography fractions to phosphorylate and activate recombinant MEK-1. Our results indicate that hepatic, EGF-sensitive MEK kinase activity may reside with a previously unidentified and physiologically relevant form of Raf and/or MEKK.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Fígado/enzimologia , MAP Quinase Quinase Quinase 1 , MAP Quinase Quinase Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-raf/metabolismo , Animais , Feminino , Feto/enzimologia , Humanos , MAP Quinase Quinase Quinase 2 , MAP Quinase Quinase Quinases/isolamento & purificação , Gravidez , Proteínas Proto-Oncogênicas c-raf/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Hepatocyte proliferation and differentiation occur simultaneously during late mammalian gestation. We hypothesized that regulation of hepatocyte growth and differentiation would be coordinated in late gestation fetal hepatocyte cultures such that proliferation would be most active in a population of less well-differentiated cells. Cultured fetal hepatocytes (embryonic d 19 and 21; E19 and E21) were studied using double staining immunofluorescent microscopy. Differentiation was assessed as staining for alpha-fetoprotein (AFP), three markers of enzymic differentiation (glucokinase [GK], phosphoenolpyruvate carboxykinase [PEPCK], and carbamoyl phosphate synthase [CPS]), and a hepatocyte cell-cell adhesion molecule (C-CAM). Proliferation was assessed using immunocytochemical detection of proliferating cell nuclear antigen (PCNA) or 5-bromo-2'-deoxy-uridine (BrdU) incorporation into DNA. Fetal hepatocyte cultures consisted of a heterogeneous population of cells, slightly more than half of which were proliferative under defined, growth factor-free conditions. These cultures were heterogeneous for AFP expression. There was no correlation between the expression of AFP and PCNA or AFP and S-phase entry (BrdU staining) during the first 48 h in culture. Similar results were obtained in staining for the enzymic differentiation markers and C-CAM. In addition, the differentiation status of cultured fetal hepatocytes was unrelated to a presumed indicator of mature growth regulation, mitogenic responsiveness to transforming growth factor alpha (TGFalpha), and hepatocyte growth factor (HGF). Finally, absence of any correlation between proliferation and differentiated phenotype was supported by in vivo studies using staining for PCNA, AFP, CPS, and PEPCK in liver sections. These results indicate that the developmental program governing differentiation of late gestation fetal rat hepatocytes is independent from mechanisms controlling proliferation.
Assuntos
Fígado/citologia , Fígado/embriologia , Animais , Animais Recém-Nascidos , Biomarcadores/análise , Moléculas de Adesão Celular/análise , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , DNA/biossíntese , Feto , Idade Gestacional , Glucoquinase/análise , Fator de Crescimento de Hepatócito/farmacologia , Fígado/efeitos dos fármacos , Fosfoenolpiruvato Carboxiquinase (GTP)/análise , Ratos , Fator de Crescimento Transformador alfa/farmacologia , alfa-Fetoproteínas/análiseRESUMO
A 16-year-old boy with insulin-dependent diabetes mellitus (IDDM) and a history of marginal glycemic control had severe hypoglycemia unawareness and a marked decrease in insulin requirement. His counterregulatory hormone response at the time of hypoglycemia suggested adrenocortical and adrenomedullary dysfunction. Further testing confirmed Addison disease. The patient's hypoglycemia unawareness was reversed by glucocorticoid replacement, although the plasma epinephrine response to hypoglycemia remained undetectable.
Assuntos
Doença de Addison/complicações , Diabetes Mellitus Tipo 1/complicações , Hipoglicemia/etiologia , Doença de Addison/sangue , Adolescente , Hormônio Adrenocorticotrópico/sangue , Glicemia , Diabetes Mellitus Tipo 1/fisiopatologia , Epinefrina/sangue , Glucocorticoides/uso terapêutico , Humanos , Hipoglicemia/tratamento farmacológico , Masculino , PercepçãoRESUMO
Insulin-like growth factor I (IGF-I) stimulates mitogenesis in proliferating 3T3-L1 preadipocytes. However, IGF-I functions to stimulate differentiation once growth arrest occurs at confluence. Epidermal growth factor (EGF) is also a potent mitogen in these cells, but inhibits differentiation of preadipocytes. We compared mitogenic signaling via the mitogen-activated protein kinase (MAPK) pathway in response to IGF-I or EGF in proliferating, growth-arrested, and differentiating 3T3-L1 cells. IGF-I stimulation of MAPK was rapid and maximal in proliferating 3T3-L1 preadipocytes, but decreased greatly in differentiating cells. EGF was more potent than IGF-I in stimulating MAPK activity in 3T3-L1 cells, and activation of MAPK was maintained in differentiating cells. These results suggest an uncoupling of MAPK activation specific to IGF-I-mediated 3T3-L1 preadipocyte differentiation. Studies of proximal signaling revealed Shc phosphorylation and Shc/Grb2 complex formation in IGF-I-treated proliferating, but not differentiating, cells. Insulin receptor substrate-1 phosphorylation after IGF-I treatment was present in proliferating, quiescent, and differentiating preadipocytes. Shc phosphorylation and Grb2 association after EGF treatment were present throughout all phases of growth. The change in IGF-I signaling via Shc phosphorylation and MAPK activity during 3T3-L1 differentiation indicates that loss of IGF-I mitogenic signaling via the MAPK pathway is part of the differentiation process.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Adipócitos/citologia , Diferenciação Celular , Fator de Crescimento Insulin-Like I/farmacologia , Mitógenos/farmacologia , Transdução de Sinais , Células 3T3 , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Divisão Celular , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Proteína Adaptadora GRB2 , Insulina/farmacologia , Cinética , Camundongos , Fosforilação , Proteínas/metabolismo , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Domínios de Homologia de srcRESUMO
Stimulation of cell proliferation by mitogens involves tyrosine phosphorylation of proteins at the cell membrane by receptor tyrosine kinases. This promotes formation of multi-protein complexes that can activate the small G-protein, Ras. Activation of Ras, in turn, leads to sequential activation of the following three serine-threonine kinases: Raf, extracellular signal-regulated kinase kinase (MEK), and members of the family of mitogen-activated protein (MAP) kinases. Prior studies have shown that intraperitoneal injection of epidermal growth factor (EGF) leads to rapid activation of hepatic MAP kinases in adult rats but not in late gestation (E19) fetal rats (Boylan, J. M., and Gruppuso, P. A. (1996) Cell Growth & Differ. 7, 1261-1269). The present studies were undertaken to determine the mechanism for this "uncoupling" of the MAP kinase pathway. E19 fetal rats and adult male rats were injected with EGF (0.5 microg/g body weight, intraperitoneally) or with saline. After 15 min, livers were removed and prepared for kinase analyses. EGF injection led to a rapid and marked activation of hepatic Raf and MEK in both fetal and adult rats, whereas MAP kinase activation was minimal in fetal as opposed to adult rats. Examination of the ontogeny of this dissociation of MAP kinase activation from MEK activation showed gradual acquisition of intact signaling as an adult hepatocyte phenotype was attained during the first 4 postnatal weeks. Over this period, MAP kinase content as determined by Western immunoblotting was constant. Recombination experiments using partially purified fetal and adult rat liver MEK and MAP kinase showed intact MAP kinase activation in vitro, indicating that neither enzyme was irreversibly altered in the fetus. In studies using primary cultures of E19 fetal rat hepatocytes, uncoupling of MAP kinase activation from MEK activation could be induced by incubation of fetal hepatocytes for 24 h with a potent fetal hepatocyte mitogen, transforming growth factor-alpha. These findings indicate that a novel negative feedback mechanism for MAP kinase regulation may be active in developing rat hepatocytes.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Feto/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Animais , Regulação para Baixo , Ativação Enzimática , Feminino , Feto/enzimologia , Fígado/embriologia , Fígado/enzimologia , Gravidez , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Late gestation fetal rat hepatocytes can proliferate under defined in vitro conditions in the absence of added mitogens. However, this capacity declines with advancing gestational age of the fetus from which the hepatocytes are derived. The present studies were undertaken to investigate this change in fetal hepatocyte growth regulation. Examination of E19 fetal hepatocyte primary cultures using immunocytochemistry for 5-bromo-2'-deoxyuridine (BrdU) incorporation showed that approximately 80% of these cells traverse S-phase of the cell cycle over the first 48 h in culture. Similarly, 65% of E19 hepatocytes maintained in culture under defined mitogen-free conditions for 24 h showed nuclear expression of proliferating cell nuclear antigen (PCNA). These in vitro findings correlated with a high level of immunoreactive PCNA in immunofluorescent analyses of E19 liver. In contrast, E21 (term) liver showed little immunoreactive PCNA. The in vivo finding was recapitulated by in vitro studies showing that E21 hepatocytes had low levels of BrdU incorporation during the first day in culture and were PCNA negative shortly after isolation. However, within 12 h of plating, E21 hepatocytes showed cytoplasmic staining for PCNA. Although maintained under mitogen-free conditions, PCNA expression progressed synchronously to a nucleolar staining pattern at 24 to 48 h in culture followed by intense, diffuse nuclear staining at 60 h which disappeared by 72 h. This apparently synchronous cell cycle progression was confirmed by studies showing peak BrdU incorporation on the third day in culture. Whereas DNA synthesis by both E19 and E21 hepatocytes was potentiated by transforming growth factor alpha (TGF alpha), considerable mitogen-independent DNA synthesis was seen in hepatocytes from both gestational ages. These results may indicate that fetal hepatocytes come under the influence of an exogenous, in vivo growth inhibitory factor as term approaches and that this effect is relieved when term fetal hepatocytes are cultured.
Assuntos
Animais Recém-Nascidos , Divisão Celular , Fígado/embriologia , Fígado/crescimento & desenvolvimento , Mitógenos/farmacologia , Animais , Núcleo Celular/química , Citoplasma/química , DNA/biossíntese , Feminino , Imunofluorescência , Idade Gestacional , Imuno-Histoquímica , Fígado/citologia , Gravidez , Antígeno Nuclear de Célula em Proliferação/análise , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador alfa/farmacologiaRESUMO
We previously isolated and characterized a coding region determinant-binding protein (CRD-BP) that might regulate c-myc mRNA post-transcriptionally. CRD-BP binds specifically to the coding region of c-myc mRNA and might stabilize c-myc mRNA in vitro by protecting it from endonucleolytic cleavage. Since c-myc abundance is regulated during embryonic development and cell replication, we investigated whether CRD-BP is also regulated in animal tissues. We focused on CRD-BP expression during rat liver development and liver regeneration, because c-myc mRNA is regulated post-transcriptionally in both cases. CRD-BP expression parallels c-myc expression during liver development; the protein is present in fetal and neonatal liver but is absent or in low abundance in adult liver. In contrast, the up-regulation of c-myc mRNA following partial hepatectomy is not accompanied by up-regulation of CRD-BP. To our knowledge, CRD-BP is the first example of a putative mammalian mRNA-binding protein that is abundant in a fetal tissue but either absent from or scarce in adult tissues. Its expression in fetal liver and in transformed cell lines suggests CRD-BP is an oncofetal protein.