Time-dependent changes in glucose and insulin regulation during intermittent hypoxia and continuous hypoxia.

Hypoxia manifests in many forms including the short repetitive intermittent hypoxia (IH) of sleep apnoea and the continuous hypoxia (CH) of altitude, both of which may impact metabolic function. Based on our own previous studies and the available literature, we hypothesized that whereas acute exposure to IH and CH would lead to comparable metabolic dysfunction, with longer-term exposure, metabolism would normalize to a greater extent with CH than IH. Studies were conducted in lean C57BL/6J mice exposed to either IH or CH for 1 day or 4 weeks and compared to either intermittent air (IA) or unhandled (UN) controls, respectively. We utilized the frequently sampled intravenous glucose tolerance test and minimal model analyses to determine insulin-dependent (insulin sensitivity; S (I)) and insulin-independent (glucose effectiveness; S (g)) glucose disposal, as well as the insulin response to glucose (acute insulin response to glucose; AIR(g)). Our data show that 1-day exposure impaired the glucose tolerance and caused reductions in S (g) and AIR(g) in both the IH and CH groups, but only IH caused a significant decrease in S (I) (7.5 ± 2.7 vs. 17.0 ± 5.3 µU ml(-1) min(-1); p < 0.05). After 4-week exposure, there was evidence of metabolic adaptation in both hypoxic groups, however, in the CH group, there was a supranormal increase in S (I) relative to both UN and IH groups. We conclude that in lean mice, the marked metabolic dysfunction that occurs with acute exposure to hypoxia is reversed to a greater extent with chronic CH exposure than chronic IH exposure.

Restoration of glucose metabolism in leptin-resistant mouse hearts after acute myocardial infarction through the activation of survival kinase pathways.

In the normal heart, leptin modulates cardiac metabolism. It is unknown, however, what effect leptin has on cardiac metabolism and outcomes in acute myocardial infarction (MI). This study was performed to test the hypothesis that leptin signaling increases glucose metabolism and attenuates injury in the acutely infarcted heart. Mice with (ObR(+/+)) and without (ObR(-/-)) cardiomyocyte specific expression of leptin receptor (ObR) were randomly assigned to experimental MI or sham procedure, and studied 3 days later. ObR(+/+) and ObR(-/-) sham mice were not significantly different in any measured outcome. However, after MI, ObR(-/-) mice had greater cardiac dysfunction, left ventricular dilation, and levels of oxidative stress. These worse indices of cardiac injury in ObR(-/-) mice were associated with attenuated signal transducer and activator of transcription (STAT) 3, phosphatidylinositol-3-kinase (PI3K), and Akt signaling, decreased malonyl CoA content, and reduced mitochondrial pyruvate dehydrogenase and electron transport Complex I, II and IV activities. Furthermore, ObR(-/-) mice maintained high rates of cardiac fatty acid oxidation after MI, whereas ObR(+/+) mice demonstrated a switch away from fatty acid oxidation to glucose metabolism. Restoration of cardiac STAT3, PI3K and Akt activity and mitochondrial function in ObR(-/-) mice post-MI was accomplished by ciliary neurotrophic factor (CNTF), an established STAT3 activator, administered immediately after MI. Moreover, CNTF therapy resulted in mitigation of cardiac structural and functional injury, attenuated levels of oxidative stress, and rescued glucose metabolism in the infarcted ObR(-/-) heart. These data demonstrate that impaired cardiac leptin signaling results in metabolic inflexibility for glucose utilization in the face of cardiac stress, and greater morbidity after MI. Further, these studies show that cardiac glucose metabolism can be restored in leptin-resistant hearts by CNTF-mediated activation of survival kinases, resulting in multiple improved structural and functional outcomes post-MI.

Activation of cardiac hypertrophic signaling pathways in a transgenic mouse with the human PRKAG2 Thr400Asn mutation.

Human mutations in PRKAG2, the gene encoding the gamma2 subunit of AMP activated protein kinase (AMPK), cause a glycogen storage cardiomyopathy. In a transgenic mouse with cardiac specific expression of the Thr400Asn mutation in PRKAG2 (TG(T400N)), we previously reported initial cardiac hypertrophy (ages 2-8 weeks) followed by dilation and failure (ages 12-20 weeks). We sought to elucidate the molecular mechanisms of cardiac hypertrophy. TG(T400N) mice showed significantly increased cardiac mass/body mass ratios up to approximately 3-fold beginning at age 2 weeks. Cardiac expression of ANP and BNP were approximately 2- and approximately 5-fold higher, respectively, in TG(T400N) relative to wildtype (WT) mice at age 2 weeks. NF-kappaB activity and nuclear translocation of the p50 subunit were increased approximately 2- to 3-fold in TG(T400N) hearts relative to WT during the hypertrophic phase. Phosphorylated Akt and p70S6K were elevated approximately 2-fold as early as age 2 weeks. To ascertain whether these changes in TG(T400N) mice were a consequence of increased AMPK activity, we crossbred TG(T400N) with TG(alpha2DN) mice, which express a dominant negative, kinase dead mutant of the AMPK alpha2 catalytic subunit and have low myocardial AMPK activity. Genetic reversal of AMPK overactivity led to a reduction in hypertrophy, nuclear translocation of NF-kappaB, phosphorylated Akt, and p70S6K. We conclude that inappropriate activation of AMPK secondary to the T400N PRKAG2 mutation is associated with the early activation of NF-kappaB and Akt signaling pathway, which mediates cardiac hypertrophy.

SGLT1, a novel cardiac glucose transporter, mediates increased glucose uptake in PRKAG2 cardiomyopathy.

Human mutations in the gene PRKAG2 encoding the gamma2 subunit of AMP-activated protein kinase (AMPK) cause a glycogen storage cardiomyopathy. Transgenic mice (TG(T400N)) with the human T400N mutation exhibit inappropriate activation of AMPK and consequent glycogen storage in the heart. Although increased glucose uptake and activation of glycogen synthesis have been documented in PRKAG2 cardiomyopathy, the mechanism of increased glucose uptake has been uncertain. Wildtype (WT), TG(T400N), and TG(alpha2DN) (carrying a dominant negative, kinase dead alpha2 catalytic subunit of AMPK) mice were studied at ages 2-8 weeks. Cardiac mRNA expression of sodium-dependent glucose transporter 1 (SGLT1), but not facilitated-diffusion glucose transporter 1 (GLUT1) or GLUT4, was increased approximately 5- to 7-fold in TG(T400N) mice relative to WT. SGLT1 protein was similarly increased at the cardiac myocyte sarcolemma in TG(T400N) mice. Phlorizin, a specific SGLT1 inhibitor, attenuated cardiac glucose uptake in TG(T400N) mice by approximately 40%, but not in WT mice. Chronic phlorizin treatment reduced cardiac glycogen content by approximately 25% in TG(T400N) mice. AICAR, an AMPK activator, increased cardiac SGLT1 mRNA expression approximately 3-fold in WT mice. Relative to TG(T400N) mice, double transgenic (TG(T400N)/TG(alpha2DN)) mice had decreased ( approximately 50%) cardiac glucose uptake and decreased (approximately 70%) cardiac SGLT1 expression. TG(T400N) hearts had increased binding activity of the transcription factors HNF-1 and Sp1 to the promoter of the gene encoding SGLT1. Our data suggest that upregulation of cardiac SGLT1 is responsible for increased cardiac glucose uptake in the TG(T400N) mouse. Increased AMPK activity leads to upregulation of SGLT1, which in turn mediates increased cardiac glucose uptake.

Extracellular superoxide dismutase regulates cardiac function and fibrosis.

Extracellular superoxide dismutase (EC-SOD) is an antioxidant that protects the heart from ischemia and the lung from inflammation and fibrosis. The role of cardiac EC-SOD under normal conditions and injury remains unclear. Cardiac toxicity, a common side effect of doxorubicin, involves oxidative stress. We hypothesize that EC-SOD is critical for normal cardiac function and protects the heart from oxidant-induced fibrosis and loss of function. C57BL/6 and EC-SOD-null mice were treated with doxorubicin, 15 mg/kg (i.p.). After 15 days, echocardiography was used to assess cardiac function. Left ventricle (LV) tissue was used to assess fibrosis and inflammation by staining, Western blot, and hydroxyproline analysis. At baseline, EC-SOD-null mice have LV wall thinning and increases in LV end diastolic dimensions compared to wild-type mice but have normal cardiac function. After doxorubicin, EC-SOD-null mice have decreases in fractional shortening not apparent in WT mice. Lack of EC-SOD also leads to increases in myocardial apoptosis and significantly more LV fibrosis and inflammatory cell infiltration. Administration of the metalloporphyrin AEOL 10150 abrogates the loss of cardiac function, and potentially fibrosis, associated with doxorubicin treatment in both wild-type and EC-SOD KO mice. EC-SOD is critical for normal cardiac morphology and protects the heart from oxidant-induced fibrosis, apoptosis, and loss of function. The antioxidant metalloporphyrin AEOL 10150 effectively protects cardiac function from doxorubicin-induced oxidative stress in vivo. These findings identify targets for the use of antioxidant agents in oxidant-induced cardiac fibrosis.

Chronic intermittent hypoxia increases left ventricular contractility in C57BL/6J mice.

Intermittent hypoxia (IH) commonly occurs in patients with obstructive sleep apnea and can cause a wide range of pathology, including reduced left ventricular (LV) ejection fraction in rats as determined by echocardiography, in rodent models. We utilized echocardiography and pressure-volume (PV) loop analyses to determine whether LV contractility was decreased in inbred C57BL/6J mice exposed to IH and whether blockade of beta-adrenergic receptors modified the response to hypoxia. Adult male 9- to 10-wk-old mice were exposed to 4 wk of IH (nadir inspired O(2) 5-6% at 60 cycles/h for 12 h during the light period) or intermittent air (IA) as control. A second group of animals were exposed to the same regimen of IH or IA, but in the presence of nonspecific beta-blockade with propranolol. Cardiac function was assessed by echocardiography and PV loop analyses, and mRNA and protein expression in ventricular homogenates was determined. Contrary to our expectations, we found with PV loop analyses that LV ejection fraction (63.4 +/- 3.5 vs. 50.5 +/- 2.6%, P = 0.015) and other measures of LV contractility were increased in IH-exposed animals compared with IA controls. There were no changes in contractile proteins, atrial natriuretic peptide levels, LV posterior wall thickness, or heart weight with IH exposure. However, cAMP levels were elevated after IH, and propranolol administration attenuated the increase in LV contractility induced by IH exposure. We conclude that, contrary to our hypothesis, 4 wk of IH exposure in C57BL/6J mice causes an increase in LV contractility that occurs independent of ventricular hypertrophy and is, in part, mediated by activation of cardiac beta-adrenergic pathways.

Leptin signalling reduces the severity of cardiac dysfunction and remodelling after chronic ischaemic injury.

AIMS: Leptin is elevated under conditions of both obesity and heart failure (HF), and activation of leptin receptor (ObR) signalling is known to increase in vivo cardiac contractility and to have anti-hypertrophic effects on the left ventricle (LV). However, it is unknown whether ObR signalling is altered in cardiomyocytes after myocardial infarction (MI) leading to HF, or if a deficiency in ObR signalling leads to worse HF. METHODS AND RESULTS: In separate experimental protocols, C57BL/6J and leptin-deficient (ob/ob) mice underwent open-chest surgery to induce permanent left coronary artery ligation (CAL) or had a sham operation. Subgroups of ob/ob mice examined were lean (food-restricted), obese (food-ad libitum), and leptin repleted. Four weeks post-surgery, cardiac structure and function was examined by echocardiography, and the activation of cardiac leptin signalling was characterized through quantitative PCR, western blotting, and DNA-binding activities. CAL produced echocardiographic evidence of HF in C57BL/6J mice, elevated circulating leptin, increased cardiomyocyte leptin and ObR expression, and activated myocardial signal transducer and activator of transcription-3 (STAT3). In leptin-deficient ob/ob mice, whether lean or obese, CAL caused increased hypertrophy and dilation, decreased contractility of the LV, and worsened survival relative to wildtype or leptin-repleted mice after CAL. In ob/ob mice, activation of cardiac STAT3 signalling after CAL is enhanced in the presence of leptin and parallels the induction of the STAT3-responsive genes, tissue-inhibitor of metalloproteinase-1 and heat shock protein-70. CONCLUSION: These data demonstrate that HF increases ObR signalling in cardiomyocytes and that activation of ObR signalling improves functional outcomes in chronic ischaemic injury leading to HF.

Identification and characterization of a response element in the EGFR promoter that mediates transcriptional repression by 1,25-dihydroxyvitamin D3 in breast cancer cells.

Reduction of epidermal growth factor receptor (EGFR) mRNA and protein by 1,25-dihydroxyvitamin D3 has been documented in MCF7, T47D, and BT549 breast cancer cells. In the present report, functional mapping of the EGFR promoter in BT549 cells has revealed a sequence of DNA between nucleotide positions -536 and -478 that resembles a consensus vitamin D response element (VDRE) and confers a vitamin D response upon both the homologous and a minimal heterologous promoter. In vitro footprinting and gel shift assays demonstrate the presence of an unidentified nuclear factor that is required for strong binding of the vitamin D receptor (VDR) to this putative VDRE. An Sp1 binding site was also identified in close proximity and shown to bind Sp1 from nuclear extract. Mutational analysis and functional studies using a minimal heterologous promoter provide evidence that the VDR in concert with an unknown nuclear partner mediates basal EGFR repression through displacement of Sp1 which is augmented in the presence of a ligand.

A high leucine diet mitigates cardiac injury and improves survival after acute myocardial infarction.

OBJECTIVE: Evidence suggests that branched chain amino acids (BCAAs) are beneficial in treating human disease. It is unknown, however, what impact BCAAs have on outcomes in acute myocardial infarction (MI). This study was performed to test the hypothesis that the specific BCAA leucine mitigates cardiac injury and improves survival post-MI. MATERIALS/METHODS: 11-12 week old male C57BL/6 mice were subjected to experimental MI or sham procedure, and provided regular chow (RC; 1.5% leucine) or a high leucine diet (HLD; 5% leucine), and followed for 3½ or 28 days. All mice were studied by echocardiography and cardiac catheterization, and all hearts were collected for histologic measurements of hypertrophy, fibrosis and apoptosis. Inflammation, hypertrophic gene expression, signal transduction, and glucose, palmitate and leucine metabolism were also measured in 3½day hearts. RESULTS: Except for increased leucine and decreased glucose oxidation, an HLD had no effect on measured outcomes in sham mice. With MI, cardiac structure, function, and survival were significantly improved with an HLD. At 3½days post-MI, an HLD increased cardiac hypertrophic signaling and decreased inflammation. Cardiac leucine oxidation was decreased in RC mice post-MI, but significantly increased with an HLD. These changes in metabolism were accompanied by a significant increase in cardiac ATP content in the HLD group. Finally, at both 3½ and 28 days post-MI, an HLD increased compensatory hypertrophy, and attenuated cardiac fibrosis and apoptosis. CONCLUSIONS: An HLD increases compensatory hypertrophy, attenuates fibrosis and apoptosis, and positively modulates oxidative metabolism to improve cardiac structure, function, and survival post-MI.

Chronic intermittent hypoxia exposure improves left ventricular contractility in transgenic mice with heart failure.

We previously reported the unexpected finding that 4 wk of exposure to intermittent hypoxia (IH), which simulates the hypoxic stress of obstructive sleep apnea, improved LV cardiac function in healthy, lean C57BL/6J mice. The purpose of the present study was to assess the impact of 4 wk of IH on cardiac function in a transgenic murine model that exhibits a natural history of heart failure. We hypothesized that IH exposure would exacerbate cardiac decompensation in heart failure. Adult male FVB (wild type) and transgenic mice with cardiac overexpression of tumor necrosis factor α (TNF-αTG) at 10-12 wk of age were exposed to 4 wk of IH (nadir inspired oxygen 5-6% at 60 cycles/h for 12 h during light period) or intermittent air (IA) as control. Cardiac function was assessed by echocardiography and pressure-volume loop analyses, and mRNA and protein expression were performed on ventricular homogenates. TNF-αTG mice exposed to IA exhibited impaired LV contractility and increased LV dilation associated with markedly elevated cardiac expression of atrial natriuretic peptide and brain natriuretic peptide compared with wild-type mice. When wild-type FVB mice were exposed to IH, they exhibited increases in arterial pressure and dP/dt(max), consistent with our previous report in C57BL/6J mice. Surprisingly, we found that TNF-αTG mice exposed to IH showed a reduction in end-diastolic volume (38.7 ± 3.8 to 22.2 ± 2.1 ul; P < 0.01) and an increase in ejection fraction (29.4 ± 2.5 to 41.9 ± 3.1%; P < 0.05). In contrast to our previous study in C56Bl/6J mice, neither FVB nor TNF-αTG mice exhibited an upregulation in ß-adrenergic expression or cAMP in response to IH exposure. We conclude that 4 wk of exposure to IH in mice induces adaptive responses that improve cardiac function in not only healthy animals but also in animals with underlying heart failure.

Cardiac-specific leptin receptor deletion exacerbates ischaemic heart failure in mice.

AIMS: the obesity-related adipokine, leptin, has multiple actions on peripheral organs, including the mitigation of adverse cardiovascular outcomes after myocardial infarction (MI). Although we recently demonstrated that leptin, its receptor, and downstream signalling are up-regulated in the heart after MI, the significance of intact cardiomyoctye leptin signalling is unknown. Therefore, our objective was to generate a cardiomyocyte-specific leptin receptor knock-out (ObRKO) mouse to determine whether worse cardiac outcomes after MI result from impaired leptin signalling in cardiomyocytes. METHODS AND RESULTS: tamoxifen-inducible ObRKO mice were subjected to experimental MI or sham surgeries and studied after 1 month. After MI, ObRKO mice displayed a loss of cardiac signal transducer and activator of transcription (STAT) 3 and adenosine monophosphate-activated protein kinase (AMPK) signalling. Worse survival and cardiac morbidity were also seen in the ObRKO mouse post-MI, including decreased contractile function and glycolytic metabolism, and increased left ventricular dilation, hypertrophy, collagen deposition, matrix metalloproteinase activity, apoptosis, and inflammation. Treatment of ObRKO mice post-MI with an ObR-independent AMPK activator improved cardiac function and restored many of these maladaptive processes to wild-type levels. CONCLUSION: these data indicate that leptin signalling mitigates cardiac injury in the post-MI failing heart by acting directly on cardiomyocytes to increase STAT3 and AMPK activation, to decrease cardiac hypertrophy, apoptosis, and inflammation, and to limit deleterious changes in cardiac structure, function, and glycolytic metabolism.

Leptin attenuates cardiac apoptosis after chronic ischaemic injury.

AIMS: We have previously shown that activation of leptin signalling in the heart reduces cardiac morbidity and mortality after myocardial infarction (MI). In the present study, we tested the hypothesis that leptin signalling limits cardiac apoptosis after MI through activation of signal transducer and activator of transcription (STAT)-3 responsive anti-apoptotic genes, including B-cell lymphoma (bcl)-2 and survivin, that serve to downregulate the activity of caspase-3. METHODS AND RESULTS: Hearts from C57BL/6J and three groups of leptin-deficient Ob/Ob mice (food-restricted, ad libitum, and leptin-repleted) were examined 4 weeks after permanent left coronary artery ligation or sham operation. Inflammatory and apoptotic cell number was determined in cardiac sections by immunostaining. Expression of cardiac bcl-2, survivin, and pro and active caspase-3 was determined and correlated with in vitro caspase-3 activity. In the absence of MI, both lean and obese leptin-deficient mice exhibited increased cardiac apoptosis compared with wild-type mice. After MI, the highest rates of apoptosis were seen in the infarcted tissue of lean and obese Ob/Ob mice. Further, leptin-deficient hearts, as well as hearts from wild-type mice treated with the STAT-3 inhibitor WP1066, exhibited blunted anti-apoptotic bcl-2 and survivin gene expression, and increased caspase-3 protein expression and activity. The increased caspase-3 activity and apoptosis in hearts of leptin-deficient mice after MI was significantly attenuated in Ob/Ob mice replete with leptin, reducing apoptosis to levels comparable to that observed in wild-type mice after MI. CONCLUSION: These results demonstrate that intact leptin signalling post-MI acts through STAT-3 to increase anti-apoptotic bcl-2 and survivin gene expression and reduces caspase-3 activity, consistent with a cardioprotective role of leptin in the setting of chronic ischaemic injury.

SGLT1 is a novel cardiac glucose transporter that is perturbed in disease states.

AIMS: Cardiac myocytes depend on a delicate balance of glucose and free fatty acids as energy sources, a balance that is disrupted in pathological states such as diabetic cardiomyopathy and myocardial ischaemia. There are two families of cellular glucose transporters: the facilitated-diffusion glucose transporters (GLUT); and the sodium-dependent glucose transporters (SGLT). It has long been thought that only the GLUT isoforms, GLUT1 and GLUT4, are responsible for cardiac myocyte glucose uptake. However, we discovered that one SGLT isoform, SGLT1, is also an important glucose transporter in heart. In this study, we aimed to determine the human and murine cardiac expression pattern of SGLT1 in health and disease and to determine its regulation. METHODS AND RESULTS: SGLT1 was largely localized to the cardiac myocyte sarcolemma. Changes in SGLT1 expression were observed in disease states in both humans and mouse models. SGLT1 expression was upregulated two- to three-fold in type 2 diabetes mellitus and myocardial ischaemia (P < 0.05). In humans with severe heart failure, functional improvement following implantation of left ventricular assist devices led to a two-fold increase in SGLT1 mRNA (P < 0.05). Acute administration of leptin to wildtype mice increased cardiac SGLT1 expression approximately seven-fold (P < 0.05). Insulin- and leptin-stimulated cardiac glucose uptake was significantly (P < 0.05) inhibited by phlorizin, a specific SGLT1 inhibitor. CONCLUSION: Our data suggest that cardiac SGLT1 expression and/or function are regulated by insulin and leptin, and are perturbed in disease. This is the first study to examine the regulation of cardiac SGLT1 expression by insulin and leptin and to determine changes in SGLT1 expression in cardiac disease.

Leptin signaling in the failing and mechanically unloaded human heart.

BACKGROUND: Increased circulating leptin is present in human heart failure, and leptin deficiency is linked to worse outcomes in chronic ischemic injury. In the present observational study, we tested the hypothesis that cardiac leptin production and signaling are increased in the failing human heart, and that mechanical unloading with a ventricular assist device (VAD) reverses these changes. METHODS AND RESULTS: All studies were performed using human cardiac tissue obtained from (1) hearts not matched for transplantation (nonfailing), (2) at the time of cardiac transplant (failing), or (3) paired samples at the time of VAD implant (pre-VAD) and removal (post-VAD). The expression of brain naturetic peptide, leptin, leptin receptor, and tumor necrosis factor alpha mRNA was measured, and the protein expression of leptin and its receptor was examined by Western blot and immunofluorescent staining of cardiac sections. The assessment of leptin signaling was performed by measuring the phosphorylation state of the leptin receptor. The phosphorylation state of signal transducer and activator of transcription-3 and AMP-activated kinase proteins were also measured. All data are expressed as mean+/-SEM with a statistical significance in failing relative to nonfailing groups determined by Student independent t test, and the significance between pre- and post-VAD groups determined by paired t test. In failing human hearts, the mRNA expressions of leptin and its receptor were increased 5.4+/-0.3-fold (P<0.05) and 4.5+/-0.3-fold (P<0.05), respectively, with similar changes in protein. The phosphorylation state of both the leptin receptor and signal transducer and activator of transcription-3 proteins were increased 1.4+/-0.1-fold (P<0.05), and the level of phosphorylated AMP-activated kinase protein was increased 1.9+/-0.2-fold (P<0.05). Mechanical unloading of the failing human heart with a VAD resulted in no change in tumor necrosis factor alpha expression but a marked decrease in leptin production to 1.7+/-0.1% (P<0.05) and leptin receptor expression to 3.0+/-0.2% (P<0.05) of pre-VAD levels. Phosphorylation of the leptin receptor, signal transducer and activator of transcription-3, and AMP-activated kinase were also decreased to 45+/-7%, 75+/-8%, and 58+/-8% of pre-VAD values, respectively (P<0.05 for all values). CONCLUSIONS: These results indicate that the failing human heart increases expression of leptin and its receptor and that mechanical unloading downregulates this increase. Further, a cardioprotective role for leptin in the failing human heart is suggested through the activation of signal transducer and activator of transcription-3 and AMP-activated kinase signaling.

Growth and EGFR regulation in breast cancer cells by vitamin D and retinoid compounds.

The effect of 1,25-dihydroxyvitamin D(3), analog C (1,25-(OH)(2)-16-en-23-yn-26,27-F(6)-vitamin D(3)), 9-cis retinoic acid, and all-trans retinoic acid on the growth and expression of EGFR in MCF7, T47D, BT474, and BT549 breast cancer cells was examined. Significant growth inhibition was noted in MCF7, T47D, and BT474 cells by 8 days of treatment, while BT549 cells showed none. MCF7, T47D, and BT549 cells treated with 1,25-dihydroxyvitamin D(3) demonstrated a 50% decrease in EGFR mRNA within 2 h which was sustained to 72 h, while BT474 cells demonstrated a 200-500% increase. EGFR protein levels correlated with these mRNA changes in BT474 and BT549 cells. Measurement of mRNA stability in vitamin D treated BT474 cells indicated that there was no change in EGFR mRNA half-life. Transfection of an EGFR promoter containing reporter plasmid demonstrated vitamin D induced changes in reporter gene activity that paralleled the changes observed in EGFR mRNA and protein. Electrophoretic mobility shift assays using a putative vitamin D response element within this region of the EGFR promoter demonstrated specific VDR binding. These results indicate that the vitamin D effect on EGFR expression in breast cancer cells has a transcriptional component likely mediated through a vitamin D responsive promoter sequence. They also suggest that growth inhibition and EGFR down-regulation by vitamin D and retinoids may be related events in some breast cancer cells, but not in all.