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1.
J Virol ; 98(5): e0041624, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38624232

RESUMO

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued, enabling the virus to escape from host immunity by changing its spike antigen, while biased toward the receptor-binding domain and N-terminal domain. Here, we isolated a novel pan-SARS-CoV-2 neutralizing antibody (which we named MO11) for even the recent dominators XBB.1.16 and EG.5.1, from a convalescent patient who had received three doses of an original mRNA COVID-19 vaccination. A cryo-electron microscopy analysis of the spike-MO11 complex at 2.3 Å atomic resolution revealed that it recognizes a conserved epitope hidden behind a glycan shield at N331 on subdomain 1 (SD1), holding both the N- and C-terminal segments comprising SD1. Our identification of MO11 unveiled the functional importance of SD1 for the spike's function, and we discuss the potential availability of a novel common epitope among the SARS-CoV-2 variants.IMPORTANCENovel severe acute respiratory syndrome coronavirus 2 variants with immune evasion ability are still repeatedly emerging, nonetheless, a part of immunity developed in responding to the antigen of earlier variants retains efficacy against recent variants irrespective of the numerous mutations. In exploration for the broadly effective antibodies, we identified a cross-neutralizing antibody, named MO11, from the B cells of the convalescent patient. MO11 targets a novel epitope in subdomain 1 (SD1) and was effective against all emerging variants including XBB.1.16 and EG.5.1. The neutralizing activity covering from D614G to EG.5.1 variants was explained by the conservation of the epitope, and it revealed the importance of the subdomain on regulating the function of the antigen for viral infection. Demonstrated identification of the neutralizing antibody that recognizes a conserved epitope implies basal contribution of such group of antibodies for prophylaxis against COVID-19.


Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/química , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Humanos , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/virologia , Epitopos/imunologia , Microscopia Crioeletrônica , Domínios Proteicos , Vacinas contra COVID-19/imunologia
2.
J Infect Public Health ; 16(7): 1064-1072, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37196370

RESUMO

BACKGROUND: Omicron variants with immune evasion have emerged, and they continue to mutate rapidly, raising concerns about the weakening of vaccine efficacy, and the very elderly populations are vulnerable to Coronavirus Disease 2019 (COVID-19). Therefore, to investigate the effect of multiple doses of mRNA vaccine for the newly emerged variants on these populations, cross-neutralizing antibody titers were examined against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants, including BQ.1.1 and XBB. METHODS: Blood samples were taken from residents at four long-term care facilities in Hyogo prefecture, Japan (median age, 91 years), after 3rd (n = 67) and 4th (n = 48) mRNA vaccinations, from April to October 2022. A live virus microneutralization assay was performed to determine the neutralizing antibody titers in participants' sera. RESULTS: After 3rd vaccination, cross-neutralizing antibody prevalence against conventional (D614G) virus, Delta, Omicron BA.2, BA.5, BA.2.75, BQ.1.1, and XBB were 100%, 97%, 81%, 51%, 67%, 4%, and 21%, respectively. After 4th vaccination, the antibody positivity rates increased to 100%, 100%, 98%, 79%, 92%, 31%, and 52%, respectively. The 4th vaccination significantly increased cross-neutralizing antibody titers against all tested variants. CONCLUSION: The positivity rates for BQ.1.1 and XBB increased after 4th vaccination, although the titer value was lower than those of BA.5 and BA.2.75. Considering the rapid mutation of viruses and the efficacy of vaccines, it may be necessary to create a system that can develop vaccines suitable for each epidemic in consideration of the epidemic of the virus.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Idoso , Idoso de 80 Anos ou mais , SARS-CoV-2/genética , COVID-19/prevenção & controle , Anticorpos Amplamente Neutralizantes , Vacinação , RNA Mensageiro , Anticorpos Antivirais
3.
J Virol ; 97(6): e0028623, 2023 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-37191569

RESUMO

We identified neutralizing monoclonal antibodies against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) variants (including Omicron variants BA.5 and BA.2.75) from individuals who received two doses of mRNA vaccination after they had been infected with the D614G virus. We named them MO1, MO2, and MO3. Among them, MO1 showed particularly high neutralizing activity against authentic variants: D614G, Delta, BA.1, BA.1.1, BA.2, BA.2.75, and BA.5. Furthermore, MO1 suppressed BA.5 infection in hamsters. A structural analysis revealed that MO1 binds to the conserved epitope of seven variants, including Omicron variants BA.5 and BA.2.75, in the receptor-binding domain of the spike protein. MO1 targets an epitope conserved among Omicron variants BA.1, BA.2, and BA.5 in a unique binding mode. Our findings confirm that D614G-derived vaccination can induce neutralizing antibodies that recognize the epitopes conserved among the SARS-CoV-2 variants. IMPORTANCE Omicron variants of SARS-CoV-2 acquired escape ability from host immunity and authorized antibody therapeutics and thereby have been spreading worldwide. We reported that patients infected with an early SARS-CoV-2 variant, D614G, and who received subsequent two-dose mRNA vaccination have high neutralizing antibody titer against Omicron lineages. It was speculated that the patients have neutralizing antibodies broadly effective against SARS-CoV-2 variants by targeting common epitopes. Here, we explored human monoclonal antibodies from B cells of the patients. One of the monoclonal antibodies, named MO1, showed high potency against broad SARS-CoV-2 variants including BA.2.75 and BA.5 variants. The results prove that monoclonal antibodies that have common neutralizing epitopes among several Omicrons were produced in patients infected with D614G and who received mRNA vaccination.


Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , COVID-19 , Epitopos , Animais , Cricetinae , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/virologia , Epitopos/imunologia , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Masculino , Feminino , Pessoa de Meia-Idade , Vacinas de mRNA
4.
PLoS One ; 17(4): e0266270, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35381036

RESUMO

Continuous appearance of SARS-CoV-2 variants and mass vaccination have been intricately influencing on the COVID-19 situation. To elucidate the current status in Japan, we analyzed totally 2,000 sera in August (n = 1,000) and December (n = 1,000) 2021 collected from individuals who underwent a health check-up. The anti-N seropositive rate were 2.1% and 3.9% in August and December 2021, respectively, demonstrating a Delta variant endemic during that time; it was approximately twofold higher than the rate based on the PCR-based diagnosis. The anti-S seropositive rate was 38.7% in August and it reached 90.8% in December, in concordance with the vaccination rate in Japan. In the December cohort, 78.7% of the sera showed neutralizing activity against the Delta variant, whereas that against the Omicron was much lower at 36.6%. These analyses revealed that effective immunity against the Delta variant was established in December 2021, however, prompt three-dose vaccination is needed to overcome Omicron's outbreak.


Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Neutralizantes , COVID-19/epidemiologia , COVID-19/prevenção & controle , Humanos , Japão/epidemiologia , Vacinação
5.
Heliyon ; 6(4): e03743, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32322728

RESUMO

Matrix Gla protein (MGP), a modulator of the BMP-SMAD signals, inhibits arterial calcification in a Glu γ-carboxylation dependent manner but the role of MGP highly expressed in a subset of bone marrow (BM) mesenchymal stem/stromal cells is unknown. Here we provide evidence that MGP might be a niche factor for both normal and malignant myelopoiesis. When mouse BM hematopoietic cells were cocultured with mitomycin C-treated BM stromal cells in the presence of anti-MGP antibody, growth of hematopoietic cells was reduced by half, and maintenance of long-term culture-initiating cells (LTC-ICs) was profoundly attenuated. Antibody-mediated blockage of MGP also inhibited growth (by a fifth) and cobblestone formation (by half) of stroma-dependent MB-1 myeloblastoma cells. MGP was undetectable in normal hematopoietic cells but was expressed in various mesenchymal cells and was aberrantly high in MB-1 cells. MGP and bone morphogenetic protein (BMP)-4 were co-induced in stromal cells cocultured with both normal hematopoietic cells and MB-1 myeloblastoma cells in an oscillating several days-periodic manner. BMP-2 was also induced in stromal cells cocultured with normal hematopoietic cells but was barely expressed when cocultured with MB-1 cells. GST-pulldown and luciferase reporter assays showed that uncarboxylated MGP interacted with BMP-4 and that anti-MGP antibody abolished this interaction. LDN-193189, a selective BMP signaling inhibitor, inhibited growth and cobblestone formation of MB-1 cells. The addition of warfarin, a selective inhibitor of vitamin K-dependent Glu γ-carboxylation, did not affect MB-1 cell growth, suggesting that uncarboxylated MGP has a biological effect in niche. These results indicate that MGP may maintain normal and malignant hematopoietic progenitor cells, possibly by modulating BMP signals independently of Glu γ-carboxylation. Aberrant MGP by leukemic cells and selective induction of BMP-4 relative to BMP-2 in stromal cells might specify malignant niche.

6.
Biochem Biophys Res Commun ; 521(3): 562-568, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31677798

RESUMO

Translin, a ubiquitous RNA/DNA-binding protein that forms a hetero-octamer together with Translin-associated factor X (TRAX), possesses endoribonuclease activity and plays a physiological role in restricting the size and differentiation of mesenchymal precursor cells. However, the precise role of Translin in epithelial cells remains unclear. Here, we show evidence that Translin restricts the growth of pubertal mammary epithelial cells. The mammary epithelia of Translin-null females exhibited retarded growth before puberty, but highly enhanced growth and DNA synthesis with increased ramification after the onset of puberty. Primary cultures of Translin-null mammary epithelial cells showed augmented DNA synthesis in a ligand-independent and ligand-enhanced manner. Translin-null ovariectomized mice implanted with slow-release estrogen pellets showed enhanced length and ramification of the mammary glands. Mammary epithelial growth was also observed in ovariectomized Translin-null mice implanted with placebo pellets. Luciferase reporter assays using embryonic fibroblasts from Translin-null mice showed unaltered estrogen receptor α function. These results indicate that Translin plays a physiological role in restricting intrinsic growth, beyond mesenchymal cells, of pubertal mammary epithelial cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Proteínas de Ligação a RNA/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Replicação do DNA , Proteínas de Ligação a DNA/genética , Células Epiteliais/metabolismo , Feminino , Deleção de Genes , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a RNA/genética , Maturidade Sexual
7.
Transcription ; 10(3): 147-156, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31135261

RESUMO

Transcriptional activation by PML-RARα, an acute promyelocytic leukemia-related oncofusion protein, requires pharmacological concentrations of all-trans retinoic acid (ATRA). However, the mechanism by which the liganded PML-RARα complex leads to the formation of the preinitiation complex has been unidentified. Here we demonstrate that the Mediator subunit MED1 plays an important role in the ATRA-dependent activation of the PML-RARα-bound promoter. Luciferase reporter assays showed that PML-RARα induced significant transcription at pharmacological doses (1 µM) of ATRA; however, this was submaximal and equivalent to the level of transcription driven by intact RARα at physiological doses (1 nM) of ATRA. Transcription depended upon the interaction of PML-RARα with the two LxxLL nuclear receptor recognition motifs of MED1, and LxxLL→LxxAA mutations led to minimal transcription. Mechanistically, MED1 interacted ATRA-dependently with the RARα portion of PML-RARα through the two LxxLL motifs of MED1. These results suggest that PML-RARα initiates ATRA-induced transcription through its interaction with MED1.


Assuntos
Subunidade 1 do Complexo Mediador/metabolismo , Proteínas de Fusão Oncogênica/agonistas , Proteínas de Fusão Oncogênica/metabolismo , Ativação Transcricional/efeitos dos fármacos , Tretinoína/farmacologia , Humanos , Subunidade 1 do Complexo Mediador/química , Ligação Proteica/efeitos dos fármacos
8.
Biochem Biophys Res Commun ; 504(1): 115-122, 2018 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-30172368

RESUMO

Translin, a highly conserved DNA/RNA binding protein that forms a hetero-octamer together with Translin-associated factor X (TRAX), possesses a broad variety of functions, including RNA processing and DNA repair. Recent studies have reported that Translin is involved in mesenchymal cell physiology. Thus, here we analyzed the intrinsic role of Translin in mesenchymal cell proliferation and differentiation. Translin-deficient E11.5 mouse embryonic fibroblasts showed enhanced growth. Translin-deficient bone marrow-derived mesenchymal stem cells showed substantial expansion in vivo and enhanced proliferation in vitro. These cells also showed enhanced osteogenic and adipocytic differentiation. Histological analyses showed adipocytic hypertrophy in various adipose tissues. Translin knockout did not affect the growth of subcutaneous white adipose tissue-derived stem cells, but enhanced adipocytic differentiation was observed in vitro. Contrary to previous reports, in vitro-fertilized Translin-null mice were not runted and exhibited normal metabolic homeostasis, indicating the fragility of these mice to environmental conditions. Together, these data suggest that Translin plays an intrinsic role in restricting mesenchymal cell proliferation and differentiation.


Assuntos
Células da Medula Óssea/citologia , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Mesenquimais/citologia , Proteínas de Ligação a RNA/metabolismo , Tecido Adiposo/citologia , Animais , Diferenciação Celular , Proliferação de Células , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteogênese
9.
Biochem Biophys Res Commun ; 478(4): 1706-12, 2016 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-27596966

RESUMO

The expression of extracellular matrix protein periostin (POSTN) was attenuated in Med1(-/-) mouse embryonic fibroblasts (MEFs), which exhibited a decreased capability to support hematopoietic progenitor cells (HPCs) in vitro. When bone marrow (BM) cells were cocultured with mitomycin C-treated Med1(+/+) MEFs, or OP-9 or MS-5 BM stromal cells, in the presence of anti-POSTN antibody, the growth of BM cells and number of long-term culture-initiating cells (LTC-ICs) were attenuated. When BM cells were cocultured with Med1(-/-) MEFs in the presence of recombinant POSTN, the growth of BM cells and the number of LTC-ICs were restored. Moreover, antibody-mediated blockage of stromal cells-derived POSTN markedly reduced the growth and cobblestone formation, a leukemic stem cell feature, of stromal cell-dependent MB-1 myeloblastoma cells. POSTN was expressed both in BM cells and variably in different BM stromal cells. Expression in the latter cells was increased by physical interaction with hematopoietic cells. The receptor for POSTN, integrin αvß3, was expressed abundantly in BM stromal cells. The addition of recombinant POSTN to BM stromal cells induced intracellular signaling downstream of integrin αvß3. These results suggest that stromal cell POSTN supports both normal HPCs and leukemia-initiating cells in vitro, at least in part, indirectly by acting on stromal cells in an autocrine or paracrine manner.


Assuntos
Moléculas de Adesão Celular/genética , Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Nicho de Células-Tronco , Animais , Western Blotting , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Células Cultivadas , Técnicas de Cocultura , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HEK293 , Humanos , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Subunidade 1 do Complexo Mediador/genética , Subunidade 1 do Complexo Mediador/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma Mieloide/genética , Sarcoma Mieloide/metabolismo , Sarcoma Mieloide/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
10.
Genes Cells ; 19(1): 28-51, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24245781

RESUMO

The MED1 subunit of the Mediator transcriptional coregulator complex coactivates GATA1 and induces erythropoiesis. Here, we show the dual mechanism of GATA1- and MED1-mediated transcription. MED1 expression levels in K562 erythroleukemia cells paralleled the levels of GATA1-targeted gene transcription and erythroid differentiation. An N-terminal fragment of MED1, MED1(1-602), which is incapable of interacting with GATA1, enhanced GATA1-targeted gene transcription and erythroid differentiation, and introduction of MED1(1-602) into Med1(-/-) mouse embryonic fibroblasts (MEFs) partially rescued GATA1-mediated transcription. The C-terminal zinc-finger domain of GATA1 interacts with the MED1(1-602)-interacting coactivator CCAR1, CoCoA and MED1(681-715). CCAR1 and CoCoA synergistically enhanced GATA1-mediated transcription from the γ-globin promoter in MEFs. Recombinant GATA1, CCAR1, CoCoA and MED1(1-602) formed a complex in vitro, and GATA1, CCAR1, CoCoA and MED1 were recruited to the γ-globin promoter in K562 cells during erythroid differentiation. Therefore, in addition to the direct interaction between GATA1 and MED1, CoCoA and CCAR1 appear to relay the GATA1 signal to MED1, and multiple modes of the GATA1-MED1 axis may help to fine-tune GATA1 function during GATA1-mediated homeostasis events.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fator de Transcrição GATA1/metabolismo , Subunidade 1 do Complexo Mediador/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , Células Cultivadas , Feminino , Fator de Transcrição GATA1/genética , Humanos , Células K562 , Masculino , Subunidade 1 do Complexo Mediador/genética , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Fatores de Transcrição , Transcrição Gênica , Ativação Transcricional , gama-Globinas/genética
11.
Biochem Biophys Res Commun ; 440(1): 125-31, 2013 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-24051090

RESUMO

FGF1 and FGF2 support hematopoietic stem and progenitor cells (HSPCs) under stress conditions. In this study, we show that fibroblast growth factor (FGF7) may be a novel niche factor for HSPC support and leukemic growth. FGF7 expression was attenuated in mouse embryonic fibroblasts (MEFs) deficient for the MED1 subunit of the Mediator transcriptional coregulator complex. When normal mouse bone marrow (BM) cells were cocultured with Med1(+/+) MEFs or BM stromal cells in the presence of anti-FGF7 antibody, the growth of BM cells and the number of long-time culture-initiating cells (LTC-ICs) decreased significantly. Anti-FGF7 antibody also attenuated the proliferation and cobblestone formation of MB1 stromal cell-dependent myeloblastoma cells. The addition of recombinant FGF7 to the coculture of BM cells and Med1(-/-) MEFs increased BM cells and LTC-ICs. FGF7 and its cognate receptor, FGFR2IIIb, were undetectable in BM cells, but MEFs and BM stromal cells expressed both. FGF7 activated downstream targets of FGFR2IIIb in Med1(+/+) and Med1(-/-) MEFs and BM stromal cells. Taken together, we propose that FGF7 supports HSPCs and leukemia-initiating cells indirectly via FGFR2IIIb expressed on stromal cells.


Assuntos
Fator 7 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Leucemia/metabolismo , Células-Tronco Mesenquimais/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Fator 7 de Crescimento de Fibroblastos/genética , Deleção de Genes , Células-Tronco Hematopoéticas/citologia , Humanos , Leucemia/genética , Subunidade 1 do Complexo Mediador/genética , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais
12.
Biochem Biophys Res Commun ; 440(1): 184-9, 2013 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-24055033

RESUMO

The MED1 subunit of the Mediator transcriptional coregulator complex is a nuclear receptor-specific coactivator. A negative feedback mechanism of thyroid-stimulating hormone (TSH, or thyrotropin) expression in the thyrotroph in the presence of triiodothyronine (T3) is employed by liganded thyroid hormone receptor ß (TRß) on the TSHß gene promoter, where conventional histone-modifying coactivators act as corepressors. We now provide evidence that MED1 is a ligand-dependent positive cofactor on this promoter. TSHß gene transcription was attenuated in MED1 mutant mice in which the nuclear receptor-binding ability of MED1 was specifically disrupted. MED1 stimulated GATA2- and Pit1-mediated TSHß gene promoter activity in a ligand-independent manner in cultured cells. MED1 also stimulated transcription from the TSHß gene promoter in a T3-dependent manner. The transcription was further enhanced when the T3-dependent corepressors SRC1, SRC2, and HDAC2 were downregulated. Hence, MED1 is a T3-dependent and -independent coactivator on the TSHß gene promoter.


Assuntos
Subunidade 1 do Complexo Mediador/metabolismo , Regiões Promotoras Genéticas , Tireotropina Subunidade beta/genética , Ativação Transcricional , Tri-Iodotironina/metabolismo , Animais , Linhagem Celular , Feminino , Fator de Transcrição GATA2/metabolismo , Humanos , Masculino , Subunidade 1 do Complexo Mediador/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Fator de Transcrição Pit-1/metabolismo
13.
Mol Cell Biol ; 32(8): 1483-95, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22331469

RESUMO

The Mediator subunit MED1 is essential for mammary gland development and lactation, whose contribution through direct interaction with estrogen receptors (ERs) is restricted to involvement in pubertal mammary gland development and luminal cell differentiation. Here, we provide evidence that the MED24-containing submodule of Mediator functionally communicates specifically with MED1 in pubertal mammary gland development. Mammary glands from MED1/MED24 double heterozygous knockout mice showed profound retardation in ductal branching during puberty, while single haploinsufficient glands developed normally. DNA synthesis of both luminal and basal cells were impaired in double mutant mice, and the expression of ER-targeted genes encoding E2F1 and cyclin D1, which promote progression through the G(1)/S phase of the cell cycle, was attenuated. Luciferase reporter assays employing double mutant mouse embryonic fibroblasts showed selective impairment in ER functions. Various breast carcinoma cell lines expressed abundant amounts of MED1, MED24, and MED30, and attenuated expression of MED1 and MED24 in breast carcinoma cells led to attenuated DNA synthesis and growth. These results indicate functional communications between the MED1 subunit and the MED24-containing submodule that mediate estrogen receptor functions and growth of both normal mammary epithelial cells and breast carcinoma cells.


Assuntos
Neoplasias da Mama/metabolismo , Glândulas Mamárias Animais , Neoplasias Mamárias Animais/metabolismo , Subunidade 1 do Complexo Mediador/metabolismo , Complexo Mediador/metabolismo , Envelhecimento/metabolismo , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ciclina D1/metabolismo , Fator de Transcrição E2F1/metabolismo , Feminino , Fase G1 , Humanos , Glândulas Mamárias Animais/embriologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Animais/patologia , Camundongos , Fase S
14.
Mol Cell Biol ; 30(20): 4818-27, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20713445

RESUMO

MED1/TRAP220, a subunit of the transcriptional Mediator/TRAP complex, is crucial for various biological events through its interaction with distinct activators, such as nuclear receptors and GATA family activators. In hematopoiesis, MED1 plays a pivotal role in optimal nuclear receptor-mediated myelomonopoiesis and GATA-1-induced erythropoiesis. In this study, we present evidence that MED1 in stromal cells is involved in supporting hematopoietic stem and/or progenitor cells (HSPCs) through osteopontin (OPN) expression. We found that the proliferation of bone marrow (BM) cells cocultured with MED1 knockout (Med1(-/-)) mouse embryonic fibroblasts (MEFs) was significantly suppressed compared to the control. Furthermore, the number of long-term culture-initiating cells (LTC-ICs) was attenuated for BM cells cocultured with Med1(-/-) MEFs. The vitamin D receptor (VDR)- and Runx2-mediated expression of OPN, as well as Mediator recruitment to the Opn promoter, was specifically attenuated in the Med1(-/-) MEFs. Addition of OPN to these MEFs restored the growth of cocultured BM cells and the number of LTC-ICs, both of which were attenuated by the addition of the anti-OPN antibody to Med1(+/+) MEFs and to BM stromal cells. Consequently, MED1 in niche appears to play an important role in supporting HSPCs by upregulating VDR- and Runx2-mediated transcription on the Opn promoter.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Subunidade 1 do Complexo Mediador/genética , Subunidade 1 do Complexo Mediador/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Células Estromais/metabolismo , Animais , Técnicas de Cocultura , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , Subunidade 1 do Complexo Mediador/deficiência , Camundongos , Camundongos Knockout , Osteopontina/deficiência , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Calcitriol/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Células Estromais/citologia , Transfecção , Técnicas do Sistema de Duplo-Híbrido
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