RESUMEN
Fibroblast growth factor (FGF) signaling can bypass the requirement for estrogen receptor (ER) activation in the growth of ER-positive (ER+) breast cancer cells. Fibroblast growth factor-1 stimulation leads to phosphorylation of the adaptor protein Suc1-associated neurotrophic factor-induced tyrosine-phosphorylated target (SNT-1) on C-terminal tyrosine residues, whereas it is constitutively bound through its N-terminal phosphotyrosine-binding domain (PTB) to FGF receptors (FGFRs). By expressing the PTB domain of SNT-1 (SNT-1 PTB) in an inducible manner in an ER+ breast carcinoma line, ML20, we asked whether we could uncouple FGFR activation from its downstream signaling components and abrogate FGF-1-induced antiestrogen-resistant growth. Induction of SNT-1 PTB resulted in a significant decrease of FGF-1-dependent tyrosine phosphorylation of endogenous SNT-1, strong inhibition of complex formation between SNT-1, Gab-1 and Sos-1, and reduced activation of Ras, mitogen-activated protein kinase (MAP kinase), and Akt. SNT-1 PTB also inhibited the phosphorylation of p70S6K on Thr421/Ser424 and Ser411, which may result from the abrogation of MAP kinase activity. Moreover, we also observed a decreased phosphorylation of the MAP kinase-independent site Thr389. This may reflect both inhibition of PI-3 kinase pathways and mammalian target of rapamycin (mTOR)-dependent signaling, as the phosphorylation of Thr389 site was sensitive to treatment with the PI3-K and mTOR inhibitors, LY294002 and rapamycin, respectively. Collectively these results suggest that SNT-1 plays a pivotal role in FGF-dependent activation of the Ras-MAP kinase, PI-3 kinase, and mTOR pathways in these cells. Fibroblast growth factor-1 dependent colony formation of ML20 cells in media containing the pure antiestrogen ICI 182,780 was also markedly inhibited upon induction of SNT-1 PTB, suggesting that blockade of FGFR-SNT-1 interactions might abrogate FGF-mediated antiestrogen resistance in breast cancers.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Neoplasias de la Mama/enzimología , Carcinoma/enzimología , Moduladores de los Receptores de Estrógeno/farmacología , Factor 1 de Crecimiento de Fibroblastos/fisiología , Proteínas de la Membrana/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosfotirosina/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/genética , Antineoplásicos/farmacología , Neoplasias de la Mama/patología , Carcinoma/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Doxiciclina/farmacología , Resistencia a Antineoplásicos/genética , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de la Membrana/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Transducción de Señal/genéticaRESUMEN
The mdr1b gene is thought to be a "stress-responsive" gene, however it is unknown if this gene is regulated by p53 in the whole animal. Moreover, it is unknown if overexpression of mdr1b affects cell survival. The dependence of mdr1b upon p53 for upregulation was evaluated in p53 knockout mice. Wild-type (wt) or p53-/- mice were treated singly or in combination with gamma irradiation (IR) and/or the potent DNA damaging agent, diethylnitrosoamine (DEN). Both IR and DEN induced mdr1b in wild-type animals, but not in the p53-/- mice. IR also upregulated endogenous mdr1b in the H35 liver cell line, and the mdr1b promoter was activated by IR and activation correlated with p53 levels; moreover activation required an intact p53 binding site. Colony survival studies revealed that co-transfection of both mdr1b and p53 dramatically reduced colony numbers compared to cells transfected with either p53 or mdr1b alone and cells microinjected with both mdr1b and p53 had a more dramatic loss in viability compared to cells injected with either expression vector alone. Further studies using acridine orange and ethidium bromide to measure apoptosis revealed that mdr1b caused apoptosis and this was enhanced by p53, however the increased apoptosis required a functional p53 transactivation domain. These studies indicate that mdr1b is a downstream target of p53 in the whole animal and expression of mdr1b facilitates p53-mediated cell death.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/efectos de la radiación , Alquilantes/farmacología , Animales , Secuencia de Bases , Sitios de Unión , Muerte Celular/fisiología , Células Cultivadas , Secuencia Conservada , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Dietilnitrosamina/farmacología , Rayos gamma , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/efectos de la radiación , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Radiación Ionizante , Ratas , Homología de Secuencia de Ácido Nucleico , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba , Irradiación Corporal Total , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
The promoter of the rat pgp2/mdr1b gene has a GC-rich region (pgp2GC) that is highly conserved in mdr genes and contains an consensus Sp1 site. Sp1's role in transactivation of the pgp2/mdr1b promoter was tested in Drosophila Schneider cells. The pgp2/mdr1b promoter was strongly activated by co-transfected wild type Sp1 but not mutant Sp1 and mutation of the Sp1 site abrogated Sp1-dependent transactivation. In gel shift assays, the same mutations abolished Sp1-DNA complex formation. Moreover, basal activity of the pgp2/mdr1b Sp1 mutant promoter was dramatically lower. Enforced ectopic overexpression of Sp1 in H35 rat hepatoma cells revealed that cell lines overexpressing Sp1 had increased endogenous pgp2/mdr1b mRNA, demonstrating that Sp1 activates the endogenous pgp2/mdr1b gene. Pgp2GC oligonucleotide also bound Egr-1 in gel shift assays and Egr-1 competitively displaced bound Sp1. In transient transfections of H35 cells (and human LS180 and HepG2 cells) Egr-1 potently and specifically suppressed pgp2/mdr1b promoter activity and mutations in the Egr-1 site decreased Egr-1 binding and correlated with pgp2/mdr1b up-regulation. Ectopic overexpression of Egr-1 in H35 cells decreased Pgp expression and selectively increased vinblastine sensitivity. In conclusion, Sp1 positively regulates while Egr-1 negatively regulates the rat pgp2/mdr1b gene. Moreover, competitive interactions between Sp1 and Egr-1 in all likelihood determine the constitutive expression of the pgp2/mdr1b gene in H35 cells.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Proteínas de Unión al ADN/metabolismo , Genes MDR , Proteínas Inmediatas-Precoces/metabolismo , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo , Dedos de Zinc , Animales , Cricetinae , ADN/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz , Regulación de la Expresión Génica , Humanos , Ratones , Regiones Promotoras Genéticas , Ratas , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Células Tumorales Cultivadas , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Loss of functional p53 paradoxically results in either increased or decreased resistance to chemotherapeutic drugs. The inconsistent relationship between p53 status and drug sensitivity may reflect p53's selective regulation of genes important to cytotoxic response of chemotherapeutic agents. We reasoned that the discrepant effects of p53 on chemotherapeutic cytotoxicity is due to p53-dependent regulation of the multidrug resistance gene (MDR1) expression in tumors that normally express MDR1. To test the hypothesis that wild-type p53 regulates the endogenous mdr1 gene we stably introduced a trans-dominant negative (TDN) p53 into rodent H35 hepatoma cells that express P-glycoprotein (Pgp) and have wild-type p53. Levels of Pgp and mdr1a mRNA were markedly elevated in cells expressing TDN p53 and were linked to impaired p53 function (both transactivation and transrepression) in these cells. Enhanced mdr1a gene expression in the TDN p53 cells was not secondary to mdr1 gene amplification and Pgp was functional as demonstrated by the decreased uptake of vinblastine. Cytotoxicity assays revealed that the TDN p53 cell lines were selectively insensitive to Pgp substrates. Sensitivity was restored by the Pgp inhibitor reserpine, demonstrating that only drug retention was the basis for loss of drug sensitivity. Similar findings were evident in human LS180 colon carcinoma cells engineered to overexpress TDN p53. Therefore, the p53 inactivation seen in cancers likely leads to selective resistance to chemotherapeutic agents because of up-regulation of MDR1 expression.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes p53 , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Regiones Promotoras Genéticas , Transfección , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
The P-glycoprotein (Pgp) reversing agent, reserpine, induces MDR1 mRNA and PGP protein in human colon carcinoma cells (Schuetz, E. G., Beck, W. T., and Schuetz, J. D. (1996) Mol. Pharmacol. 49, 311-318) and in H35 rat hepatoma cells. Reserpine's interference with cellular dopamine utilization suggested that dopamine and dopaminergics might be important physiological regulators of PGP expression. Initial studies demonstrated that the H35 cells express the D2 dopamine receptor. Pgp protein and pgp2/mdr1b mRNA was increased (maximum of 10- and 8-fold, respectively) by the potent D2 dopamine receptor agonists bromocriptine, R(-)-propylnorapomorphine hydrochloride, and quinpirole, and Pgp protein induction was blocked by D2 receptor antagonists spiperone and clozapine. D2 receptor agonist induction of pgp2/mdr1b mRNA was paralleled by transcriptional activation of the pgp2/mdr1b promoter but blocked by pretreatment with the D2 dopamine receptor antagonists, spiperone, eticlopride, and clozapine. Co-transfection of a D2 dopamine receptor expression vector enhanced bromocriptine's transcriptional activation of the pgp2/mdr1b promoter. The G-protein, Galphai2, is required for bromocriptine transcriptional activation because the G-protein inhibitor, pertussis toxin, suppressed bromocriptine's activation of pgp2/mdr1b transcription and co-transfection of a dominant negative Galphai2 abrogated bromocriptine activation of pgp2/mdr1b. Gi proteins can transduce signals by activation of mitogen-activated protein kinases (MAPKs), and because Raf-1 is a known activator of MDR1, we tested for Raf-1 involvement. Co-transfection of a dominant negative Raf-1 failed to block bromocriptine induction of pgp2/mdr1b, and bromocriptine treatment caused no phosphorylation of the MAP kinase kinase substrates p42 and p44, demonstrating that the MAP kinase pathway was not involved. These are the first studies demonstrating transcriptional activation of an MDR gene by dopamine receptor agonists and that this activation occurs by a signal transduction pathway requiring the D2 dopamine receptor coupled to a functional G-protein.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Bromocriptina/farmacología , Agonistas de Dopamina/farmacología , Resistencia a Múltiples Medicamentos/genética , Genes MDR , Regulación hacia Arriba , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Secuencia de Aminoácidos , Animales , Antagonistas de Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Proteínas de Unión al GTP/metabolismo , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-raf , Ratas , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Transducción de Señal , Transcripción Genética , Células Tumorales CultivadasRESUMEN
The human liver cytochromes P450 3A (CYP3As), orthologous to the rat glucocorticoid inducible forms, are composed of at least four differentially expressed members. To begin the study of the molecular events in the glucocorticoid regulation of CYP3A5, we fused 5' sequences of CYP3A5 to the chloramphenicol acetyltransferase gene in a vector that contains the herpes simplex virus thymidine kinase promoter. In HepG2 cells, the largest 5' CYP3A5 gene fragment (1.4 kb) suppressed the TK promoter. However, suppression was overcome by addition of 10 microM dexamethasone. A series of unidirectional deletions revealed a unique 219-bp fragment (-891 to -1109 bp upstream from the transcriptional start site) that conferred dexamethasone responsiveness on the TK promoter regardless of either the distance or orientation from the promoter and thus appears to be an enhancer. Nucleotide sequence analysis of this CYP3A5 enhancer revealed no consensus 15-bp glucocorticoid responsive element (GRE) (GGTACANNNTGTTCT); however, two GRE "half-sites" (TGTTCT) were found separated by 160 bp. Although dexamethasone stimulated the CYP3A5 enhancer only 3-4-fold in HepG2 cells, the CYP3A5 enhancer was stimulated 7- and 12-fold in immortalized primary human hepatocytes and primary rat hepatocyte cultures, respectively. The glucocorticoid receptor (GCR) seems to be indispensable to this process because 1) dexamethasone induction can be blocked by the antiglucocorticoid RU-486, 2) dexamethasone-dependent transcriptional activation of the CYP3A5 enhancer in HepG2 cells required cotransfection of an expression vector containing the intact GCR, yet 3) cotransfection with a plasmid that contains a mutation in the ligand binding domain of the GCR does not activate the CYP3A5 enhancer in the presence of dexamethasone. To further localize the dexamethasone responsive region of the 219-bp CYP3A5 enhancer, it was subdivided and fused to the TKCAT expression vector. Transfection analysis in HepG2 cells demonstrated that neither GRE half-site can independently confer dexamethasone responsiveness on the TK promoter. Block mutations of either of the two GRE half-sites or point mutations at specific GCR binding sites eliminates dexamethasone inducibility, demonstrating the half-sites need to interact. Electromobility shift assays indicate that the CYP3A5 5'-GRE half-site 1) specifically binds purified GCR, 2) can displace binding of the GCR to a consensus GRE, and 3) shifts a protein in HepG2 nuclear extracts that is supershifted by GCR antibody, demonstrating that this enhancer is an authentic GRE. This is the first study to demonstrate that a member of the human CYP3A gene family contains an enhancer that binds the GCR and that this binding is critical to transcriptional activation by dexamethasone.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Dexametasona/farmacología , Elementos de Facilitación Genéticos , Hígado/enzimología , Oxigenasas de Función Mixta/genética , Animales , Secuencia de Bases , Citocromo P-450 CYP2E1 , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/metabolismo , ADN , Activación Enzimática , Humanos , Hígado/citología , Virus del Tumor Mamario del Ratón/genética , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Ratas , Células Tumorales CultivadasRESUMEN
We investigated whether the glucocorticoid-mediated mechanisms controlling P-glycoprotein (pgp2 or mdr1b) are similar in normal hepatocytes compared with the H35 hepatoma cell line. In primary rat hepatocytes, dexamethasone (DEX) caused a dose- and time-dependent decrease in the amount of the pgp2 mRNA, which correlated with functional pgp2 expression (intracellular accumulation of [3H]vincristine). The suppression of pgp2 mRNA was specific for glucocorticoids because a representative estrogen and progestin were without effect, and DEX suppression of pgp2 mRNA could be reversed by cotreatment with an anti-glucocorticoid. DEX suppression of pgp2 mRNA appears to be posttranscriptional because following actinomycin D inhibition of new RNA synthesis, the pgp2 transcript disappeared at a faster rate in DEX treated versus untreated hepatocytes. Moreover, transcriptional activity of chloramphenicol acetyltransferase plasmids containing the pgp2 promoter in primary rat hepatocytes was unaffected by DEX treatment. Thus, suppression of pgp2 mRNA by glucocorticoids in primary hepatocytes is due to a decrease in pgp2 mRNA stability. In contrast, in the H35 hepatoma cell line, DEX dose dependently increased pgp protein and pgp2 mRNA, effects which parallel transcriptional activation of the pgp2 promoter. Activation of the pgp2 promoter was specific for glucocorticoids since a representative estrogen had no significant effect on transcription of the pgp2 promoter and RU486 blocked DEX activation of pgp2 transcription. Transcriptional activation of the pgp2 promoter was not due to a global up-regulation of basal transcription factors because DEX treatment did not activate either a herpes simplex virus thymidine kinase promoter or the SV40 early gene promoter. Further studies with a panel of pgp2 5' sequence deletion plasmids revealed that the minimal promoter (-66 bp) was not activated by DEX. In contrast, inclusion of sequences up to -177 bp restored DEX-dependent transcriptional activation. These are the first studies to demonstrate that glucocorticoids regulate pgp2 by different mechanisms in normal rat hepatocytes compared to the H35 hepatoma cell line.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Dexametasona/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Neoplasias Hepáticas Experimentales/metabolismo , Hígado/metabolismo , Animales , Secuencia de Bases , Células Cultivadas , Estradiol/farmacología , Glucocorticoides/antagonistas & inhibidores , Hígado/citología , Hígado/efectos de los fármacos , Neoplasias Hepáticas Experimentales/genética , Mifepristona/farmacología , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/metabolismo , Ratas , Activación Transcripcional , Células Tumorales Cultivadas , Tirosina Transaminasa/metabolismo , Vincristina/biosíntesisRESUMEN
The effects of left ventricular hypertrophy induced by hyperthyroidism on three biochemical markers of parasympathetic innervation were investigated. In response to subcutaneous injections of thyroxine (400 micrograms/kg; T4) for 6 days, the left ventricle, but not the right, developed significant hypertrophy (20%). In the enlarged left ventricle, acetylcholine (ACh) content and choline acetyltransferase (ChAT) activity per chamber were elevated approx. 25-30%, although no change in these two markers was evident when the data were expressed per unit wet weight. Immunoblot analysis showed that the relative abundance of ChAT protein increased in the hypertrophied left ventricle in correlation with the increased ChAT activity. No changes in ACh content, ChAT activity and ChAT relative abundance were evident in the right ventricle of T4-treated animals. Although hyperthyroidism did not alter AChE specific activity (per unit wet weight) in the left ventricle, the percent activities of the individual AChE globular forms were affected in this chamber. Specifically, T4-treatment reduced the percent activity of globular (G)4 AChE by 20% and increased that of the combined G1 and G2 AChE pool by 15%. Interestingly, in the hypertrophied left ventricle total AChE activity in its extracellular or functionally-relevant pool was reduced due to a loss of G4 AChE activity. These results show that a compensatory increase in parasympathetic innervation can occur during hyperthyroid-induced left ventricular hypertrophy. However, the reduced activity of the functionally-relevant AChE pool suggests that the clearance of ACh after release may be slowed in the hypertrophied left ventricle.
Asunto(s)
Acetilcolina/metabolismo , Acetilcolinesterasa/metabolismo , Cardiomegalia/metabolismo , Colina O-Acetiltransferasa/metabolismo , Hipertiroidismo/complicaciones , Tiroxina , Animales , Cardiomegalia/etiología , Hipertiroidismo/inducido químicamente , Immunoblotting , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The organochlorine compound mirex (dodecachloro-octahydro-1,3,4-metheno-2H-cyclobuta-CD- pentalene) induces an adaptive liver growth dependent on the hormonal status of the experimental animal. In the intact laboratory rat, mirex induces liver growth that is an expression of both cellular hyperplasia and hypertrophy. However, in rats subjected to adrenalectomy, mirex induces liver growth that is essentially hyperplastic. Corticosterone supplements given to rats subjected to adrenalectomy and treated with mirex restore the hypertrophic component of liver growth. Therefore it appears that the expression of the hypertrophic component of mirex-induced liver growth is corticosterone dependent. To further explore the hormonal modulation of the expression of mirex-induced adaptive liver growth, rats subjected to thyroidectomy were studied. In male rats subjected to thyroidectomy, a single oral dose of mirex (100 mg/kg body wt) increased relative liver weight (liver wt/body wt x 100) by 62% within 72-hr after mirex administration. Liver growth occurred in the absence of [3H]thymidine incorporation into liver DNA. Thus the observed liver growth was totally hypertrophic. However, in mirex-dosed rats subjected to thyroidectomy given twice-daily subcutaneous injections of thyroxine (5 mg/kg body wt), relative liver weight was increased by 204% of the control value within 72-hr after mirex administration, and there was a peak of [3H]thymidine incorporation into liver DNA 54 hr after mirex administration. These studies suggest that the expression of hyperplasia in mirex-induced adaptive liver growth is thyroxine dependent.
Asunto(s)
Hígado/efectos de los fármacos , Hígado/crecimiento & desarrollo , Mírex/farmacología , Tiroidectomía , Animales , ADN/análisis , Hipertrofia , Hígado/química , Hígado/patología , Masculino , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Endogámicas , Tiroxina/administración & dosificaciónRESUMEN
Mirex, an organochlorine pesticide, is a potent inducer of liver hyperplasia and hypertrophy in intact (INT) rats. In contrast, mirex elicits predominantly hyperplastic liver growth in adrenalectomized (ADX) rats and hypertrophic liver growth in thyroidectomized (THX) rats. Supplements of glucocorticoids restore liver hypertrophy and inhibit DNA synthesis in ADX mirex-dosed rats. Because responsiveness to glucocorticoids is in part dependent on the number and affinity of glucocorticoid receptors (GR) we have measured hepatic GR levels and dexamethasone inducibility of tyrosine aminotransferase (TAT) in mirex-dosed INT, ADX, and THX rats. Specific [3H]dexamethasone binding sites decreased to 48, 49, and 59% of control values in mirex-dosed INT, ADX, and THX rats, respectively, with no changes in the apparent equilibrium dissociation constant at 48 h postdose. The significant depletion of hepatic cytosolic GR in all experimental groups dosed with mirex failed to impair both uninduced and dexamethasone-induced TAT levels in these groups. It appears that the decrease in steroid binding is neither a result of an interaction between mirex and hepatic GR nor a simple "dilution" of receptors due to the accompanying liver hypertrophy. Taken collectively, these data suggest that despite the ligand-independent downregulation of hepatic GR, responsiveness of hepatocytes from mixed-dosed rats to glucocorticoids is not altered. Therefore, glucocorticoid-mediated liver hypertrophy in mirex-dosed rats probably involves either 1) modulation of steps subsequent to ligand binding and activation translocation of hepatic GR, 2) glucocorticoid effects at extrahepatic sites that release factors that are the actual effectors of parenchymal cell enlargement, or 3) an unconventional receptor-mediated mechanism.