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1.
JAMA ; 319(19): 2021-2031, 2018 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-29800214

RESUMEN

Importance: Individuals with adenomatous polyps are advised to undergo repeated colonoscopy surveillance to prevent subsequent colorectal cancer (CRC), but the relationship between adenomas at colonoscopy and long-term CRC incidence is unclear. Objective: To compare long-term CRC incidence by colonoscopy adenoma findings. Design, Setting, and Participants: Multicenter, prospective cohort study of participants in the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer randomized clinical trial of flexible sigmoidoscopy (FSG) beginning in 1993 with follow-up for CRC incidence to 2013 across the United States. Participants included 154 900 men and women aged 55 to 74 years enrolled in PLCO of whom 15 935 underwent colonoscopy following their first positive FSG screening result. The final day of follow-up was December 31, 2013. Exposures: Enrolled participants had been randomized to FSG or usual care. Participants who underwent FSG and had abnormal findings were referred for follow-up. Subsequent colonoscopy findings were categorized as advanced adenoma (≥1 cm, high-grade dysplasia, or tubulovillous or villous histology), nonadvanced adenoma (<1 cm without advanced histology), or no adenoma. Main Outcomes and Measures: The primary outcome was CRC incidence within 15 years of the baseline colonoscopy. The secondary outcome was CRC mortality. Results: There were 15 935 participants who underwent colonoscopy (men, 59.7%; white, 90.7%; median age, 64 y [IQR, 61-68]). On initial colonoscopy, 2882 participants (18.1%) had an advanced adenoma, 5068 participants (31.8%) had a nonadvanced adenoma, and 7985 participants (50.1%) had no adenoma; median follow-up for CRC incidence was 12.9 years. CRC incidence rates per 10 000 person-years of observation were 20.0 (95% CI, 15.3-24.7; n = 70) for advanced adenoma, 9.1 (95% CI, 6.7-11.5; n = 55) for nonadvanced adenoma, and 7.5 (95% CI, 5.8-9.7; n = 71) for no adenoma. Participants with advanced adenoma were significantly more likely to develop CRC compared with participants with no adenoma (rate ratio [RR], 2.7 [95% CI, 1.9-3.7]; P < .001). There was no significant difference in CRC risk between participants with nonadvanced adenoma compared with no adenoma (RR, 1.2 [95% CI, 0.8-1.7]; P = .30). Compared with participants with no adenoma, those with advanced adenoma were at significantly increased risk of CRC death (RR, 2.6 [95% CI, 1.2-5.7], P = .01), but mortality risk in participants with nonadvanced adenoma was not significantly different (RR, 1.2 [95% CI, 0.5-2.7], P = .68). Conclusions and Relevance: Over a median of 13 years of follow-up, participants with an advanced adenoma at diagnostic colonoscopy prompted by a positive flexible sigmoidoscopy result were at significantly increased risk of developing colorectal cancer compared with those with no adenoma. Identification of nonadvanced adenoma may not be associated with increased colorectal cancer risk. Trial Registration: clinicaltrials.gov Identifier: NCT00002540.


Asunto(s)
Adenoma/complicaciones , Neoplasias del Colon/complicaciones , Colonoscopía , Neoplasias Colorrectales/epidemiología , Adenoma/diagnóstico , Adenoma/patología , Anciano , Estudios de Cohortes , Neoplasias del Colon/diagnóstico , Neoplasias del Colon/patología , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/mortalidad , Femenino , Estudios de Seguimiento , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Riesgo , Sigmoidoscopía
2.
Cancer ; 123(24): 4815-4822, 2017 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-28976536

RESUMEN

BACKGROUND: Screening for colorectal cancer (CRC) with flexible sigmoidoscopy (FS) has been shown to reduce CRC mortality. The current study examined whether the observed mortality reduction was due primarily to the prevention of incident CRC via removal of adenomatous polyps or to the early detection of cancer and improved survival. METHODS: The Prostate, Lung, Colorectal, and Ovarian (PLCO) cancer screening trial randomized 154,900 men and women aged 55 to 74 years. Individuals underwent FS screening at baseline and at 3 or 5 years versus usual care. CRC-specific survival was analyzed using Kaplan-Meier curves and proportional hazards modeling. The authors estimated the percentage of CRC deaths averted by early detection versus primary prevention using a model that applied intervention arm survival rates to CRC cases in the usual-care arm and vice versa. RESULTS: A total of 1008 cases of CRC in the intervention arm and 1291 cases of CRC in the usual-care arm were observed. Through 13 years of follow-up, there was no significant difference noted between the trial arms with regard to CRC-specific survival for all CRC (68% in the intervention arm vs 65% in the usual-care arm; P =.16) or proximal CRC (68% vs 62%, respectively; P = .11) cases; however, survival in distal CRC cases was found to be higher in the intervention arm compared with the usual-care arm (77% vs 66%; P<.0001). Within each arm, symptom-detected cases had significantly worse survival compared with screen-detected cases. Overall, approximately 29% to 35% of averted CRC deaths were estimated to be due to early detection and 65% to 71% were estimated to be due to primary prevention. CONCLUSIONS: CRC-specific survival was similar across arms in the PLCO trial, suggesting a limited role for early detection in preventing CRC deaths. Modeling suggested that approximately two-thirds of avoided deaths were due to primary prevention. Future CRC screening guidelines should emphasize primary prevention via the identification and removal of precursor lesions. Cancer 2017;123:4815-22. © 2017 American Cancer Society.


Asunto(s)
Detección Precoz del Cáncer/métodos , Neoplasias del Colon Sigmoide/diagnóstico , Neoplasias del Colon Sigmoide/mortalidad , Sigmoidoscopios/estadística & datos numéricos , Sigmoidoscopía/métodos , Anciano , Causas de Muerte , Supervivencia sin Enfermedad , Tecnología de Fibra Óptica , Humanos , Tamizaje Masivo/métodos , Persona de Mediana Edad , Prevención Primaria/métodos , Pronóstico , Medición de Riesgo , Neoplasias del Colon Sigmoide/prevención & control , Análisis de Supervivencia , Resultado del Tratamiento
3.
Biochim Biophys Acta ; 1843(11): 2365-75, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24946135

RESUMEN

Wnt5a and 1α,25(OH)2D3 are important regulators of endochondral ossification. In osteoblasts and growth plate chondrocytes, 1α,25(OH)2D3 initiates rapid effects via its membrane-associated receptor protein disulfide isomerase A3 (Pdia3) in caveolae, activating phospholipase A2 (PLA2)-activating protein (PLAA), calcium/calmodulin-dependent protein kinase II (CaMKII), and PLA2, resulting in protein kinase C (PKC) activation. Wnt5a initiates its calcium-dependent effects via intracellular calcium release, activating PKC and CaMKII. We investigated the requirement for components of the Pdia3 receptor complex in Wnt5a calcium-dependent signaling. We determined that Wnt5a signals through a CaMKII/PLA2/PGE2/PKC cascade. Silencing or blocking Pdia3, PLAA, or vitamin D receptor (VDR), and inhibition of calmodulin (CaM), CaMKII, or PLA2 inhibited Wnt5a-induced PKC activity. Wnt5a activated PKC in caveolin-1-silenced cells, but methyl-beta-cyclodextrin reduced its stimulatory effect. 1α,25(OH)2D3 reduced stimulatory effects of Wnt5a on PKC in a dose-dependent manner. In contrast, Wnt5a had a biphasic effect on 1α,25(OH)2D3-stimulated PKC activation; 50ng/ml Wnt5a caused a 2-fold increase in 1α,25(OH)2D3-stimulated PKC but higher Wnt5a concentrations reduced 1α,25(OH)2D3-stimulated PKC activation. Western blots showed that Wnt receptors Frizzled2 (FZD2) and Frizzled5 (FZD5), and receptor tyrosine kinase-like orphan receptor 2 (ROR2) were localized to caveolae. Blocking ROR2, but not FZD2 or FZD5, abolished the stimulatory effects of 1α,25(OH)2D3 on PKC and CaMKII. 1α,25(OH)2D3 membrane receptor complex components (Pdia3, PLAA, caveolin-1, CaM) interacted with Wnt5a receptors/co-receptors (ROR2, FZD2, FZD5) in immunoprecipitation studies, interactions that changed with either 1α,25(OH)2D3 or Wnt5a treatment. This study demonstrates that 1α,25(OH)2D3 and Wnt5a mediate their effects via similar receptor components and suggests that these pathways may interact.

4.
Connect Tissue Res ; 55 Suppl 1: 125-8, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25158196

RESUMEN

1α,25-Dihydroxy vitamin D3 [1α,25(OH)2D3] regulates growth zone chondrocytes (GC) via classical steroid hormone receptor-mediated gene transcription and by initiating rapid membrane-mediated signaling pathways. 1α,25(OH)2D3 initiates its membrane effects via its specific membrane-associated receptor (Pdia3) in caveolae. 1α,25(OH)2D3 binding to Pdia3 leads to phospholipase-A2 (PLA2)-activating protein (PLAA) activation, stimulating PLA2, resulting in prostaglandin E2 (PGE2) release and protein kinase C activation. Recently, we reported that 1α,25(OH)2D3 rapidly activates Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in GC cells. However, the roles of Pdia3, PLAA and caveolae in 1α,25(OH)2D3-dependent rapid activation of CaMKII are not clear. The aim of the present study was to evaluate the roles of Pdia3, PLAA and caveolae in 1α,25(OH)2D3 membrane-stimulated CaMKII activation. Pre-treating chondrocytes from the growth zone of the rat costochondral cartilage with antibodies against PLAA or Pdia3 blocked activation of CaMKII by 1α,25(OH)2D3. PLAA peptide rapidly activated CaMKII in GC cells. Caveolae disruption abolished CaMKII activation in response to 1α,25(OH)2D3 or PLAA peptide treatment. Immunoprecipitation studies showed increased CaM binding to PLAA in response to 1α,25(OH)2D3. The results indicated that Pdia3, PLAA and caveolae are required for rapid 1α,25(OH)2D3 membrane-mediated activation of CaMKII. 1α,25(OH)2D3 signaling via Pdia3 receptor triggered the interaction between PLAA and CaM suggesting that CaM may play a major role linking PLAA to CaMKII in membrane-mediated actions of 1α,25(OH)2D3.


Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Caveolas/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Proteína Disulfuro Isomerasas/metabolismo , Proteínas/metabolismo , Vitamina D/análogos & derivados , Animales , Caveolas/metabolismo , Células Cultivadas , Condrocitos/metabolismo , Placa de Crecimiento/efectos de los fármacos , Masculino , Osteoblastos/metabolismo , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Vitamina D/farmacología
6.
JNCI Cancer Spectr ; 2(3): pky042, 2018 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-31360863

RESUMEN

The Lung Screening Study was a multicenter controlled feasibility trial that randomly assigned subjects to undergo two rounds of screening with either low-dose spiral computed tomography (LDCT) or chest X-ray (CXR). Long-term follow-up was performed to evaluate any differences in lung-cancer-specific and all-cause mortality between arms. In 2000, subjects were randomly assigned at six screening centers. Linkage with the National Death Index was performed to ascertain long-term mortality for subjects. Median follow-up for mortality of the 1660 and 1658 subjects randomly assigned to LDCT and CXR, respectively, was 5.2 years. There were 32 and 26 deaths from lung cancer in the two groups, respectively, corresponding to lung cancer death rates of 3.84 and 3.10 per 1000 person-years, and a risk ratio of 1.24 (95% confidence interval = 0.74 to 2.08). The risk ratio for all-cause mortality was 1.20 (95% confidence interval = 0.94 to 1.54). These findings can contribute to the overall knowledge on LDCT lung cancer screening.

7.
JNCI Cancer Spectr ; 2(2): 004, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-31360843

RESUMEN

BACKGROUND: Financial hardships experienced by cancer survivors have become a prominent public health issue in the United States. Few studies of financial hardship have assessed financial holdings, including assets, debts, and their values, associated with a cancer history. METHODS: Using the 2008-2011 Medical Expenditure Panel Survey, we identified 1603 cancer survivors and 34 915 individuals age 18-64 years without a cancer history to assess associations between self-reported cancer history and assets, debts, and net worth. Distributions of self-reported asset and debt ownership, their values, and net worth were compared for adults with and without a cancer history with chi-square statistics. Multivariable ordered probit regression analysis was conducted to assess the association between cancer history and net worth using a two-sided Wald test. All analyses were stratified by age group (18-34, 35-44, 45-54, and 55-64 years). Statistical tests were two-sided. RESULTS: Among those age 45-54 years, cancer survivors had a lower proportion of home ownership than individuals without a cancer history (59.0% vs 67.1%, P = .0014) and were statistically significantly more likely to have negative net worth (≤-$3000) and less likely to have positive net worth (≥$3000). Cancer survivors were more likely to have debt than individuals without a cancer history, especially among those age 18-34 years (41.3% vs 27.1%, P < .001). CONCLUSIONS: Cancer history is associated with lower asset ownership, more debt, and lower net worth, especially in survivors age 45-54 years. Longitudinal studies of financial holdings will be important to inform development of interventions to reduce financial hardship.

8.
J Med Screen ; 24(4): 220-222, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-27903809

RESUMEN

Objective To provide evidence about the performance characteristics and consequences of bimanual ovarian palpation. Setting and methods The Prostate, Lung, Colorectal and Ovarian cancer screening trial randomized 154,900 individuals to either an intervention or control arm. Enrolled eligible participants were aged 55-74, had no history of trial cancers, and no current treatment for cancer. Intervention arm women received CA-125 tests and transvaginal ultrasound. Bimanual ovarian palpation was offered annually during the first four years of the trial. Bimanual ovarian palpation-specific sensitivity and specificity were calculated, as were rates of diagnostic procedures and resulting complications following positive bimanual ovarian palpation screens. Results A total of 20,872 women received at least one bimanual ovarian palpation, with 50,498 total bimanual ovarian palpation examinations performed. The sensitivity and specificity of bimanual ovarian palpation were 5.1% (2/39) and 99.0% (49,957/50,459), respectively; no cases were detected by bimanual ovarian palpation alone. Rates for most follow-up procedures for abnormal results in women without ovarian cancer were higher among the group with another screening test positive, except for pelvic exam, where rates were similar. No complications were reported in the bimanual ovarian palpation-only positive group. Conclusion Low sensitivity of bimanual ovarian palpation alone and in combination with other tests argue against using bimanual ovarian palpation as a screening test for ovarian cancer in asymptomatic women.


Asunto(s)
Detección Precoz del Cáncer/métodos , Neoplasias Ováricas/diagnóstico , Palpación/métodos , Anciano , Biomarcadores de Tumor/análisis , Antígeno Ca-125/análisis , Detección Precoz del Cáncer/efectos adversos , Reacciones Falso Positivas , Femenino , Humanos , Masculino , Tamizaje Masivo/efectos adversos , Tamizaje Masivo/métodos , Persona de Mediana Edad , Palpación/efectos adversos , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Ultrasonografía
9.
PLoS One ; 12(1): e0170312, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28095466

RESUMEN

Stem cell fate has been linked to the mechanical properties of their underlying substrate, affecting mechanoreceptors and ultimately leading to downstream biological response. Studies have used polymers to mimic the stiffness of extracellular matrix as well as of individual tissues and shown mesenchymal stem cells (MSCs) could be directed along specific lineages. In this study, we examined the role of stiffness in MSC differentiation to two closely related cell phenotypes: osteoblast and chondrocyte. We prepared four methyl acrylate/methyl methacrylate (MA/MMA) polymer surfaces with elastic moduli ranging from 0.1 MPa to 310 MPa by altering monomer concentration. MSCs were cultured in media without exogenous growth factors and their biological responses were compared to committed chondrocytes and osteoblasts. Both chondrogenic and osteogenic markers were elevated when MSCs were grown on substrates with stiffness <10 MPa. Like chondrocytes, MSCs on lower stiffness substrates showed elevated expression of ACAN, SOX9, and COL2 and proteoglycan content; COMP was elevated in MSCs but reduced in chondrocytes. Substrate stiffness altered levels of RUNX2 mRNA, alkaline phosphatase specific activity, osteocalcin, and osteoprotegerin in osteoblasts, decreasing levels on the least stiff substrate. Expression of integrin subunits α1, α2, α5, αv, ß1, and ß3 changed in a stiffness- and cell type-dependent manner. Silencing of integrin subunit beta 1 (ITGB1) in MSCs abolished both osteoblastic and chondrogenic differentiation in response to substrate stiffness. Our results suggest that substrate stiffness is an important mediator of osteoblastic and chondrogenic differentiation, and integrin ß1 plays a pivotal role in this process.


Asunto(s)
Diferenciación Celular , Condrocitos/citología , Condrogénesis/fisiología , Matriz Extracelular/química , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Osteogénesis/fisiología , Proliferación Celular , Células Cultivadas , Condrocitos/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/metabolismo
10.
J Steroid Biochem Mol Biol ; 147: 81-4, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25448737

RESUMEN

The secosteroid 1α,25-dihydroxy vitamin D3 [1α,25(OH)2D3] acts on cells via classical steroid hormone receptor-mediated gene transcription and by initiating rapid membrane-mediated signaling pathways. In its membrane-initiated pathway, after 1α,25(OH)2D3 interacts with protein disulfide isomerase, family A, member 3 (Pdia3) in caveolae, phospholipase A2 (PLA2) and protein kinase C (PKC) are activated. Recent efforts to determine the signaling proteins involved in the 1α,25(OH)2D3 signal from Pdia3 to PLA2 have indicated that phospholipase A2 activating protein (PLAA) and Ca(2+)/calmodulin-dependent kinase II (CaMKII) are required. PLAA is located in caveolae, where it interacts with Pdia3 and caveolin-1 (Cav-1) to initiate rapid signaling via CaMKII, activating PLA2, leading to activation of protein kinase C (PKC) and PKC-dependent responses.


Asunto(s)
Calcitriol/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Membrana Celular/metabolismo , Activación Enzimática , Silenciador del Gen , Humanos , Fosfolipasas A2/metabolismo , Proteína Quinasa C/metabolismo , Proteínas/genética , Transducción de Señal
11.
J Steroid Biochem Mol Biol ; 145: 65-74, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25263660

RESUMEN

1α,25(OH)2D3 regulates osteoblasts and chondrocytes via its membrane-associated receptor, protein disulfide isomerase A3 (Pdia3) in caveolae. 1α,25(OH)2D3 binding to Pdia3 leads to phospholipase-A2 (PLA2)-activating protein (PLAA) activation, stimulating cytosolic PLA2 and resulting in prostaglandin E2 (PGE2) release and PKCα activation, subsequently stimulating differentiation. However, how PLAA transmits the signal to cPLA2 is unknown. Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) activation is required for PLA2 activation in vascular smooth muscle cells, suggesting a similar role in 1α,25(OH)2D3-dependent signaling. The aim of the present study is to evaluate the roles of CaM and CaMKII as mediators of 1α,25(OH)2D3-stimulated PLAA-dependent activation of cPLA2 and PKCα, and downstream biological effects. The results indicated that 1α,25(OH)2D3 and PLAA-peptide increased CaMKII activity within 9 min. Silencing Cav-1, Pdia3 or Plaa in osteoblasts suppressed this effect. Similarly, antibodies against Plaa or Pdia3 blocked 1α,25(OH)2D3-dependent CaMKII. Caveolae disruption abolished activation of CaMKII by 1α,25(OH)2D3 or PLAA. CaMKII-specific and CaM-specific inhibitors reduced cPLA2 and PKC activities, PGE2 release and osteoblast maturation markers in response to 1α,25(OH)2D3. Camk2a-silenced but not Camk2b-silenced osteoblasts showed comparable effects. Immunoprecipitation showed increased interaction of CaM and PLAA in response to 1α,25(OH)2D3. The results indicate that membrane actions of 1α,25(OH)2D3 via Pdia3 triggered the interaction between PLAA and CaM, leading to dissociation of CaM from caveolae, activation of CaMKII, and downstream PLA2 activation, and suggest that CaMKII plays a major role in membrane-mediated actions of 1α,25(OH)2D3.


Asunto(s)
Huesos/metabolismo , Calcitriol/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Cartílago/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica , Fosfolipasas A2/metabolismo , Células 3T3 , Fosfatasa Alcalina/metabolismo , Alprostadil/análogos & derivados , Alprostadil/metabolismo , Animales , Membrana Celular/metabolismo , Condrocitos/citología , Masculino , Ratones , Osteoblastos/citología , Osteopontina/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Vitamina D/metabolismo
12.
J Steroid Biochem Mol Biol ; 152: 84-8, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25845934

RESUMEN

Wnt5a and 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] regulate endochondral ossification. 1α,25(OH)2D3 initiates its calcium-dependent effects via its membrane-associated receptor, protein disulfide isomerase A3 (Pdia3). 1α,25(OH)2D3 binding to Pdia3 triggers the interaction between Pdia3 and phospholipase A2 (PLA2)-activating protein (PLAA), resulting in downstream activation of calcium/calmodulin-dependent protein kinase II (CaMKII), PLA2, and protein kinase C (PKC). Wnt5a initiates its calcium-dependent effects via binding its receptors Frizzled2 (FZD2) and Frizzled5 (FZD5) and receptor tyrosine kinase-like orphan receptor 2 (ROR2), activating intracellular calcium release and stimulating PKC and CaMKII. Recent efforts to determine the inter-relation between Wnt5a and 1α,25(OH)2D3 signaling pathways have demonstrated that Wnt5a signals through a CaMKII/PLA2/PGE2/PKC cascade in chondrocytes and osteoblasts in which the components of the Pdia3 receptor complex were required. Furthermore, ROR2, but not FZD2 or FZD5, was required to mediate the calcium-dependent actions of 1α,25(OH)2D3. This review provides evidence that 1α,25(OH)2D3 and Wnt5a mediate their calcium-dependent pathways via similar receptor components and proposes that these pathways may interact since they are competing for the same receptor complex components.


Asunto(s)
Calcitriol/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/fisiología , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Línea Celular , Receptores Frizzled/metabolismo , Humanos , Ratones , Proteína Quinasa C/metabolismo , Proteínas/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Proteína Wnt-5a
13.
Steroids ; 81: 81-7, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24291576

RESUMEN

1α,25-Dihydroxy vitamin D3 [1α,25(OH)2D3] acts on cells via classical steroid hormone receptor-mediated gene transcription and by initiating rapid membrane-mediated signaling pathways. Two receptors have been implicated to play roles in 1α,25(OH)2D3 mediated rapid signaling, the classical nuclear vitamin D receptor (VDR) and protein disulfide isomerase, family A, member 3 (Pdia3). Long term efforts to investigate the roles of these two receptors demonstrated thatPdia3 is located in caveolae, where it interacts with phospholipase A2 (PLA2) activating protein (PLAA) and caveolin-1 (Cav-1) to initiate rapid signaling via Ca(++)/calmodulin-dependent protein kinase II (CaMKII), PLA2, phospholipase C (PLC), protein kinase C (PKC), and ultimately the ERK1/2 family of mitogen activated protein kinases (MAPK). VDR is present on the plasma membrane, and it is required for 1α,25(OH)2D3 induced rapid activation of Src. PDIA3+/- mice demonstrate an impaired musculoskeletal phenotype. Moreover, our studies examining mineralization of pre-osteoblasts in 3D culture have shown the physiological importance of Pdia3 and VDR interaction: knockdown of Pdia3 or VDR is characterized by impaired mineralization of the constructs.


Asunto(s)
Calcificación Fisiológica/fisiología , Membrana Celular/metabolismo , Sistema Musculoesquelético/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Receptores de Calcitriol/metabolismo , Vitamina D/análogos & derivados , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , Caveolas/metabolismo , Caveolina 1/metabolismo , Membrana Celular/efectos de los fármacos , Humanos , Ratones , Proteína Disulfuro Isomerasas/genética , Transducción de Señal/efectos de los fármacos , Fosfolipasas de Tipo C/metabolismo , Vitamina D/metabolismo , Vitamina D/farmacología
14.
Cell Signal ; 25(12): 2362-73, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23896121

RESUMEN

1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3) regulates osteoblasts through genomic and rapid membrane-mediated responses. Here we examined the interaction of protein disulfide isomerase family A, member 3 (Pdia3) and the traditional vitamin D receptor (VDR) in plasma membrane-associated responses to 1α,25(OH)2D3. We found that Pdia3 co-localized with VDR and the caveolae scaffolding protein, caveolin-1 on the surface of MC3T3-E1 osteoblasts. Immunoprecipitation showed that both Pdia3 and VDR interacted with caveolin-1. Pdia3 further interacted with phospholipase A2 activating protein (PLAA), whereas VDR interacted with c-Src. 1α,25(OH)2D3 changed the interactions and transport of the two receptors and rapidly activated phospholipase A2 (PLA2) and c-Src. Silencing either receptor or caveolin-1 inhibited both PLA2 and c-Src, indicating that the two receptors function interdependently. These two receptor dependent rapid responses to 1α,25(OH)2D3 regulated gene expression, proliferation and apoptosis of MC3T3-E1 cells. These data demonstrate the importance of both receptors and caveolin-1 in mediating membrane responses to 1α,25(OH)2D3 and subsequently regulating osteoblast biology.


Asunto(s)
Osteoblastos/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Receptores de Calcitriol/metabolismo , Vitamina D/análogos & derivados , Células 3T3 , Animales , Proteína Tirosina Quinasa CSK , Caveolina 1/metabolismo , Membrana Celular/metabolismo , Ratones , Osteoblastos/citología , Proteínas/metabolismo , Transducción de Señal , Vitamina D/metabolismo , Familia-src Quinasas/metabolismo
15.
Mol Endocrinol ; 27(7): 1065-77, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23660595

RESUMEN

Protein disulfide isomerase family A, member 3 (Pdia3) mediates many of the plasma membrane (PM)-associated rapid responses to 1α,25-dihydroxyvitamin D3 (1α,25[OH]2D3). It is not well understood how Pdia3, which is an endoplasmic reticulum (ER) chaperone, functions as a PM receptor for 1α,25(OH)2D3. We mutated 3 amino acids (K214 and R282 in the calreticulin interaction site and C406 in the isomerase catalytic site), which are important for Pdia3's ER chaperone function, and examined their role in responses to 1α,25(OH)2D3. Pdia3 constructs with and without the ER retention signal KDEL were used to investigate the PM requirement for Pdia3. Finally, we determined whether palmitoylation and/or myristoylation were required for Pdia3-mediated responses to 1α,25(OH)2D3. Overexpressing the Pdia3 R282A mutant in MC3T3-E1 cells increased PM phospholipase A2-activating protein, Rous sarcoma oncogene (c-Src), and caveolin-1 but blocked increases in 1α,25(OH)2D3-stimulated protein kinase C (PKC) seen in cells overexpressing wild-type Pdia3 (Pdia3Ovr cells). Cells overexpressing Pdia3 with K214A and C406S mutations had PKC activity comparable to untreated controls, indicating that the native response to 1α,25(OH)2D3 also was blocked. Overexpressing Pdia3[-KDEL] increased PM localization and augmented baseline PKC, but the stimulatory effect of 1α,25(OH)2D3 was comparable to that seen in wild-type cultures. In contrast, 1α,25(OH)2D3 increased prostaglandin E2 in Pdia3[±KDEL] cells. Although neither palmitoylation nor myristoylation was required for PM association of Pdia3, myristoylation was needed for PKC activation. These data indicate that both the chaperone functional domains and the subcellular location of Pdia3 control rapid membrane responses to 1α,25(OH)2D3.


Asunto(s)
Membrana Celular/metabolismo , Chaperonas Moleculares/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Vitamina D/análogos & derivados , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Membrana Celular/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Lipoilación/efectos de los fármacos , Ratones , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/metabolismo , Ácidos Mirísticos/farmacología , Unión Proteica/efectos de los fármacos , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/genética , Señales de Clasificación de Proteína , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Tunicamicina/farmacología , Vitamina D/farmacología
16.
J Steroid Biochem Mol Biol ; 132(1-2): 48-56, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22484374

RESUMEN

1α,25-Dihydroxyvitamin D(3) (1,25D3) regulates musculoskeletal cells via two different mechanisms: vitamin D receptor (VDR)-dependent gene transcription and rapid membrane-signaling via VDR as well as protein disulfide isomerase, family A, member 3 (Pdia3). In chondrocytes from the costochondral cartilage growth zone (GC), ligand binding to Pdia3 causes a rapid increase in phospholipase A(2) (PLA(2)) activity leading to release of arachidonic acid and formation of lysophospholipid (LPL). LPL activates phospholipase C (PLC), and resulting inositol trisphosphate (IP(3)) and diacylglycerol contribute to PKCα activation and downstream activation of ERK1/2. PLA(2) activating protein (PLAA) is increased in the growth zone of rat growth plates suggesting that it mediates the 1,25D3-dependent pathway. This study examined the role of PLAA in mediating 1,25D3-dependent PKC activation using GC cells and MC3T3-E1 wild-type and PLAA-silenced osteoblasts as models. PLAA, Pdia3, and caveolin-1 (Cav-1) were detected in plasma membranes and caveolae of GC and MC3T3-E1 cells. Pdia3-immunoprecipitated samples were positive for PLAA only after 1,25D3 treatment. Cav-1 was detected when immunoprecipitated with anti-Pdia3 and anti-PLAA in both vehicle and 1,25D3 treated cells. These observations were confirmed by immunohistochemistry. 1,25D3 failed to activate PLA(2) and PKC or cause PGE(2) release in PLAA-silenced cells. PLAA-antibody successfully blocked the PLAA protein and consequently suppressed PKC activity in GC and MC3T3-E1 cells. Crosslinking studies confirmed the localization of PLAA on the extracellular face on the plasma membrane in untreated MC3T3-E1 cells. Taken together, our results suggest that PLAA is an important mediator of 1α,25(OH)(2)D(3) rapid membrane mediated signaling. 1α,25(OH)(2)D(3) likely causes conformational changes bringing Pdia3 into proximity with PLAA, and aiding in transducing the signal from caveolae to the plasma membrane.


Asunto(s)
Proteína Disulfuro Isomerasas/metabolismo , Proteína Quinasa C/metabolismo , Proteínas/metabolismo , Vitamina D/análogos & derivados , Animales , Caveolina 1/genética , Caveolina 1/metabolismo , Línea Celular , Membrana Celular/metabolismo , Condrocitos/metabolismo , Dinoprostona/metabolismo , Silenciador del Gen , Ratones , Osteoblastos/metabolismo , Fosfolipasas A2/metabolismo , Proteína Disulfuro Isomerasas/genética , Proteínas/genética , ARN Interferente Pequeño/genética , Vitamina D/metabolismo
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