c-Src Inhibition Improves Cardiovascular Function but not Remodeling or Fibrosis in Angiotensin II-Induced Hypertension.

c-Src plays an important role in angiotensin II (Ang II) signaling. Whether this member of the Src family kinases is involved in the development of Ang II-induced hypertension and associated cardiovascular damage in vivo remains unknown. Here, we studied Ang II-infused (400 ng/kg/min) mice in which c-Src was partially deleted (c-Src+/-) and in wild-type (WT, c-Src+/+) mice treated with a c-Src inhibitor (CGP077675; 25 mg/kg/d). Ang II increased blood pressure and induced endothelial dysfunction in WT mice, responses that were ameliorated in c-Src+/- and CGP077675-treated mice. Vascular wall thickness and cross-sectional area were similarly increased by Ang II in WT and c-Src+/- mice. CGP077675 further increased cross-sectional area in hypertensive mice. Cardiac dysfunction (ejection fraction and fractional shortening) in Ang II-infused WT mice was normalized in c-Src+/- mice. Increased oxidative stress (plasma thiobarbituric acid-reactive substances, hydrogen peroxide, and vascular superoxide generation) in Ang II-infused WT mice was attenuated in c-Src-deficient and CGP077675-treated mice. Hyperactivation of vascular c-Src, ERK1/2 (extracellular signal-regulated kinase 1/2), and JNK (c-Jun N-terminal kinase) in hypertensive mice was normalized in CGP077675-treated and c-Src+/- mice. Vascular fibronectin was increased by Ang II in all groups and further augmented by CGP077675. Cardiac fibrosis and inflammation induced by Ang II were amplified in c-Src+/- and CGP-treated mice. Our data indicate that although c-Src downregulation attenuates development of hypertension, improves endothelial and cardiac function, reduces oxidative stress, and normalizes vascular signaling, it has little beneficial effect on fibrosis. These findings suggest a divergent role for c-Src in Ang II-dependent hypertension, where c-Src may be more important in regulating redox-sensitive cardiac and vascular function than fibrosis and remodeling.

Differential renal effects of candesartan at high and ultra-high doses in diabetic mice-potential role of the ACE2/AT2R/Mas axis.

High doses of Ang II receptor (AT1R) blockers (ARBs) are renoprotective in diabetes. Underlying mechanisms remain unclear. We evaluated whether high/ultra-high doses of candesartan (ARB) up-regulate angiotensin-converting enzyme 2 (ACE2)/Ang II type 2 receptor (AT2R)/Mas receptor [protective axis of the of the renin-angiotensin system (RAS)] in diabetic mice. Systolic blood pressure (SBP), albuminuria and expression/activity of RAS components were assessed in diabetic db/db and control db/+ mice treated with increasing candesartan doses (intermediate, 1 mg/kg/d; high, 5 mg/kg/d; ultra-high, 25 and 75 mg/kg/d; 4 weeks). Lower doses candesartan did not influence SBP, but ultra-high doses reduced SBP in both groups. Plasma glucose and albuminuria were increased in db/db compared with db/+ mice. In diabetic mice treated with intermediate dose candesartan, renal tubular damage and albuminuria were ameliorated and expression of ACE2, AT2R and Mas and activity of ACE2 were increased, effects associated with reduced ERK1/2 phosphorylation, decreased fibrosis and renal protection. Ultra-high doses did not influence the ACE2/AT2R/Mas axis and promoted renal injury with increased renal ERK1/2 activation and exaggerated fibronectin expression in db/db mice. Our study demonstrates dose-related effects of candesartan in diabetic nephropathy: intermediate-high dose candesartan is renoprotective, whereas ultra-high dose candesartan induces renal damage. Molecular processes associated with these effects involve differential modulation of the ACE2/AT2R/Mas axis: intermediate-high dose candesartan up-regulating RAS protective components and attenuating pro-fibrotic processes, and ultra-high doses having opposite effects. These findings suggest novel mechanisms through the protective RAS axis, whereby candesartan may ameliorate diabetic nephropathy. Our findings also highlight potential injurious renal effects of ultra-high dose candesartan in diabetes.

Renoprotective Effects of Atorvastatin in Diabetic Mice: Downregulation of RhoA and Upregulation of Akt/GSK3.

Potential benefits of statins in the treatment of chronic kidney disease beyond lipid-lowering effects have been described. However, molecular mechanisms involved in renoprotective actions of statins have not been fully elucidated. We questioned whether statins influence development of diabetic nephropathy through reactive oxygen species, RhoA and Akt/GSK3 pathway, known to be important in renal pathology. Diabetic mice (db/db) and their control counterparts (db/+) were treated with atorvastatin (10 mg/Kg/day, p.o., for 2 weeks). Diabetes-associated renal injury was characterized by albuminuria (albumin:creatinine ratio, db/+: 3.2 ± 0.6 vs. db/db: 12.5 ± 3.1*; *P<0.05), increased glomerular/mesangial surface area, and kidney hypertrophy. Renal injury was attenuated in atorvastatin-treated db/db mice. Increased ROS generation in the renal cortex of db/db mice was also inhibited by atorvastatin. ERK1/2 phosphorylation was increased in the renal cortex of db/db mice. Increased renal expression of Nox4 and proliferating cell nuclear antigen, observed in db/db mice, were abrogated by statin treatment. Atorvastatin also upregulated Akt/GSK3ß phosphorylation in the renal cortex of db/db mice. Our findings suggest that atorvastatin attenuates diabetes-associated renal injury by reducing ROS generation, RhoA activity and normalizing Akt/GSK3ß signaling pathways. The present study provides some new insights into molecular mechanisms whereby statins may protect against renal injury in diabetes.

Adipocyte-Specific Mineralocorticoid Receptor Overexpression in Mice Is Associated With Metabolic Syndrome and Vascular Dysfunction: Role of Redox-Sensitive PKG-1 and Rho Kinase.

Mineralocorticoid receptor (MR) expression is increased in adipose tissue from obese individuals and animals. We previously demonstrated that adipocyte-MR overexpression (Adipo-MROE) in mice is associated with metabolic changes. Whether adipocyte MR directly influences vascular function in these mice is unknown. We tested this hypothesis in resistant mesenteric arteries from Adipo-MROE mice using myography and in cultured adipocytes. Molecular mechanisms were probed in vessels/vascular smooth muscle cells and adipose tissue/adipocytes and focused on redox-sensitive pathways, Rho kinase activity, and protein kinase G type-1 (PKG-1) signaling. Adipo-MROE versus control-MR mice exhibited reduced vascular contractility, associated with increased generation of adipocyte-derived hydrogen peroxide, activation of vascular redox-sensitive PKG-1, and downregulation of Rho kinase activity. Associated with these vascular changes was increased elastin content in Adipo-MROE. Inhibition of PKG-1 with Rp-8-Br-PET-cGMPS normalized vascular contractility in Adipo-MROE. In the presence of adipocyte-conditioned culture medium, anticontractile effects of the adipose tissue were lost in Adipo-MROE mice but not in control-MR mice. In conclusion, adipocyte-MR upregulation leads to impaired contractility with preserved endothelial function and normal blood pressure. Increased elasticity may contribute to hypocontractility. We also identify functional cross talk between adipocyte MR and arteries and describe novel mechanisms involving redox-sensitive PKG-1 and Rho kinase. Our results suggest that adipose tissue from Adipo-MROE secrete vasoactive factors that preferentially influence vascular smooth muscle cells rather than endothelial cells. Our findings may be important in obesity/adiposity where adipocyte-MR expression/signaling is amplified and vascular risk increased.

Transient Receptor Potential Melastatin 7 Cation Channel Kinase: New Player in Angiotensin II-Induced Hypertension.

Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein comprising a magnesium (Mg(2+))/cation channel and a kinase domain. We previously demonstrated that vasoactive agents regulate vascular TRPM7. Whether TRPM7 plays a role in the pathophysiology of hypertension and associated cardiovascular dysfunction is unknown. We studied TRPM7 kinase-deficient mice (TRPM7Δkinase; heterozygous for TRPM7 kinase) and wild-type (WT) mice infused with angiotensin II (Ang II; 400 ng/kg per minute, 4 weeks). TRPM7 kinase expression was lower in heart and aorta from TRPM7Δkinase versus WT mice, effects that were further reduced by Ang II infusion. Plasma Mg(2+) was lower in TRPM7Δkinase versus WT mice in basal and stimulated conditions. Ang II increased blood pressure in both strains with exaggerated responses in TRPM7Δkinase versus WT groups (P<0.05). Acetylcholine-induced vasorelaxation was reduced in Ang II-infused TRPM7Δkinase mice, an effect associated with Akt and endothelial nitric oxide synthase downregulation. Vascular cell adhesion molecule-1 expression was increased in Ang II-infused TRPM7 kinase-deficient mice. TRPM7 kinase targets, calpain, and annexin-1, were activated by Ang II in WT but not in TRPM7Δkinase mice. Echocardiographic and histopathologic analysis demonstrated cardiac hypertrophy and left ventricular dysfunction in Ang II-treated groups. In TRPM7 kinase-deficient mice, Ang II-induced cardiac functional and structural effects were amplified compared with WT counterparts. Our data demonstrate that in TRPM7Δkinase mice, Ang II-induced hypertension is exaggerated, cardiac remodeling and left ventricular dysfunction are amplified, and endothelial function is impaired. These processes are associated with hypomagnesemia, blunted TRPM7 kinase expression/signaling, endothelial nitric oxide synthase downregulation, and proinflammatory vascular responses. Our findings identify TRPM7 kinase as a novel player in Ang II-induced hypertension and associated vascular and target organ damage.

Vascular injury in diabetic db/db mice is ameliorated by atorvastatin: role of Rac1/2-sensitive Nox-dependent pathways.

Oxidative stress [increased bioavailability of reactive oxygen species (ROS)] plays a role in the endothelial dysfunction and vascular inflammation, which underlie vascular damage in diabetes. Statins are cholesterol-lowering drugs that are vasoprotective in diabetes through unknown mechanisms. We tested the hypothesis that atorvastatin decreases NADPH oxidase (Nox)-derived ROS generation and associated vascular injury in diabetes. Lepr(db)/Lepr(db) (db/db) mice, a model of Type 2 diabetes and control Lepr(db)/Lepr(+) (db/+) mice were administered atorvastatin (10 mg/kg per day, 2 weeks). Atorvastatin improved glucose tolerance in db/db mice. Systemic and vascular oxidative stress in db/db mice, characterized by increased plasma TBARS (thiobarbituric acid-reactive substances) levels and exaggerated vascular Nox-derived ROS generation respectively, were inhibited by atorvastatin. Cytosol-to-membrane translocation of the Nox regulatory subunit p47(phox) and the small GTPase Rac1/2 was increased in vessels from db/db mice compared with db/+ mice, an effect blunted by atorvastatin. The increase in vascular Nox1/2/4 expression and increased phosphorylation of redox-sensitive mitogen-activated protein kinases (MAPKs) was abrogated by atorvastatin in db/db mice. Pro-inflammatory signalling (decreased IκB-α and increased NF-κB p50 expression, increased NF-κB p65 phosphorylation) and associated vascular inflammation [vascular cell adhesion molecule-1 (VCAM-1) expression and vascular monocyte adhesion], which were increased in aortas of db/db mice, were blunted by atorvastatin. Impaired acetylcholine (Ach)- and insulin (INS)-induced vasorelaxation in db/db mice was normalized by atorvastatin. Our results demonstrate that, in diabetic mice, atorvastatin decreases vascular oxidative stress and inflammation and ameliorates vascular injury through processes involving decreased activation of Rac1/2 and Nox. These findings elucidate redox-sensitive and Rac1/2-dependent mechanisms whereby statins protect against vascular injury in diabetes.

TSH signaling pathways that regulate MCP-1 in human differentiated adipocytes.

OBJECTIVE: Adipose tissue is an extra-thyroidal thyroid-stimulating hormone (TSH) target. Increases in lipolysis and in expression and release of interleukin-6 (IL-6) occur in TSH-stimulated adipocytes, and levels of circulating free fatty acids and IL-6 rise following TSH administration to patients with previous thyroidectomy and radioablation for thyroid cancer. Our first objective was to compare how TSH stimulates protein kinase A (PKA) and inhibitor of κB (IκB) kinase (IKK)-ß. Our second objective was to investigate whether TSH induces other cytokines besides IL-6. METHODS: TSH stimulation of either CHO cells expressing human TSH receptor or human abdominal subcutaneous differentiated adipocytes. RESULTS: Signaling studies showed TSH increased NADPH oxidase activity, and either diphenyleneiodonium (oxidase inhibitor) or N-acetyl cysteine (scavenger of reactive oxygen species) reduced IKKß phosphorylation. Phosphorylation of protein kinase C-δ, an upstream regulator of NADPH oxidase, was increased by TSH, and rottlerin (PKCδ inhibitor) reduced TSH-stimulated IKKß phosphorylation. TSH upregulated monocyte chemoattractant protein-1 (MCP-1) mRNA expression and the release of MCP-1 protein in human abdominal differentiated adipocytes. H89 (PKA inhibitor) and sc-514 (IKKß inhibitor) each blocked TSH-stimulated MCP-1 mRNA expression and protein release, suggesting PKA and IKKß participate in this pathway. CONCLUSIONS: These data provide new information about TSH signaling in human differentiated adipocytes, and add to the evidence that TSH is a pro-inflammatory stimulus of adipocytes.

Aldosterone signaling through transient receptor potential melastatin 7 cation channel (TRPM7) and its α-kinase domain.

We demonstrated a role for the Mg(2+) transporter TRPM7, a bifunctional protein with channel and α-kinase domains, in aldosterone signaling. Molecular mechanisms underlying this are elusive. Here we investigated the function of TRPM7 and its α-kinase domain on Mg(2+) and pro-inflammatory signaling by aldosterone. Kidney cells (HEK-293) expressing wild-type human TRPM7 (WThTRPM7) or constructs in which the α-kinase domain was deleted (ΔKinase) or rendered inactive with a point mutation in the ATP binding site of the α-kinase domain (K1648R) were studied. Aldosterone rapidly increased [Mg(2+)]i and stimulated NADPH oxidase-derived generation of reactive oxygen species (ROS) in WT hTRPM7 and TRPM7 kinase dead mutant cells. Translocation of annexin-1 and calpain-II and spectrin cleavage (calpain target) were increased by aldosterone in WT hTRPM7 cells but not in α-kinase-deficient cells. Aldosterone stimulated phosphorylation of MAP kinases and increased expression of pro-inflammatory mediators ICAM-1, Cox-2 and PAI-1 in Δkinase and K1648R cells, effects that were inhibited by eplerenone (mineralocorticoid receptor (MR) blocker). 2-APB, a TRPM7 channel inhibitor, abrogated aldosterone-induced Mg(2+) responses in WT hTRPM7 and mutant cells. In 2-APB-treated ΔKinase and K1648R cells, aldosterone-stimulated inflammatory responses were unchanged. These data indicate that aldosterone stimulates Mg(2+) influx and ROS production in a TRPM7-sensitive, kinase-insensitive manner, whereas activation of annexin-1 requires the TRPM7 kinase domain. Moreover TRPM7 α-kinase modulates inflammatory signaling by aldosterone in a TRPM7 channel/Mg(2+)-independent manner. Our findings identify novel mechanisms for non-genomic actions of aldosterone involving differential signaling through MR-activated TRPM7 channel and α-kinase.

Sphingosine-1-phosphate-induced inflammation involves receptor tyrosine kinase transactivation in vascular cells: upregulation in hypertension.

Sphingosine-1-phosphate (S1P), a multifunctional phospholipid, regulates vascular cell function. Whether S1P influences vascular inflammatory responses, particularly in hypertension, is unclear. We tested the hypothesis that S1P is a proinflammatory mediator signaling through receptor tyrosine kinase transactivation and that responses are amplified in vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats (SHRSPs), a model in which we demonstrated Edg1 (S1P1 receptor) to be a candidate gene for salt-sensitive hypertension. Vascular smooth muscle cell from Wistar-Kyoto rats and SHRSPs were studied. S1P receptor subtypes, S1P1 and S1P2, were similarly expressed in Wistar-Kyoto rats and SHRSPs. S1P induced phosphorylation of epidermal growth factor receptor and platelet-derived growth factor and activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, with amplified effects in SHRSPs versus Wistar-Kyoto rats. Inhibition of epidermal growth factor receptor and platelet-derived growth factor (with AG1478 and AG1296, respectively) abolished S1P-induced phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase in Wistar-Kyoto rats with variable effects in SHRSPs. Vascular smooth muscle cell inflammation was evaluated by expression of adhesion molecules and functional responses assessed by monocyte adhesion. S1P stimulated expression of intercellular adhesion molecule 1 and vascular cell adhesion protein 1 and promoted monocyte adhesion, particularly in SHRSP cells. S1P-mediated inflammation was blunted by AG1478 and AG1296 in SHRSP cells. VPC23019, a S1P1 receptor antagonist, inhibited S1P-induced mitogen-activated protein kinase phosphorylation, intercellular adhesion molecule 1 and vascular cell adhesion protein 1 expression, and monocyte adhesion. Our data indicate that molecular processes underlying vascular inflammation and cell adhesion in SHRSPs involve S1P/S1P1 receptors and phosphorylation of receptor tyrosine kinases. We identify a novel pathway linking S1P/S1P1 receptors to specific proinflammatory signaling pathways through epidermal growth factor receptor and platelet-derived growth factor transactivation, a process that is upregulated in SHRSPs. Such molecular events may contribute to vascular inflammation in hypertension.

Transient receptor potential melastatin 7 (TRPM7) cation channels, magnesium and the vascular system in hypertension.

Decreased Mg(2+) concentration has been implicated in altered vascular reactivity, endothelial dysfunction and structural remodeling, processes important in vascular changes and target organ damage associated with hypertension. Unlike our knowledge of other major cations, mechanisms regulating cellular Mg(2+) handling are poorly understood. Until recently little was known about protein transporters controlling transmembrane Mg(2+) influx. However, new research has uncovered a number of genes and proteins identified as transmembrane Mg(2+) transporters, particularly transient receptor potential melastatin (TRPM) cation channels, TRPM6 and TRPM7. Whereas TRPM6 is found primarily in epithelial cells, TRPM7 is ubiquitously expressed. Vascular TRPM7 has been implicated as a signaling kinase involved in vascular smooth muscle cell growth, apoptosis, adhesion, contraction, cytoskeletal organization and migration, and is modulated by vasoactive agents, pressure, stretch and osmotic changes. Emerging evidence suggests that vascular TRPM7 function might be altered in hypertension. The present review discusses the importance of Mg(2+) in vascular biology in hypertension and focuses on transport systems, mainly TRPM7, that might play a role in the control of vascular Mg(2+) homeostasis. Elucidation of the relationship between the complex systems responsible for regulation of Mg(2+) homeostasis, the role of TRPM7 in vascular signaling, and the cardiovascular impact will be important for understanding the clinical implications of hypomagnesemia in cardiovascular disease.

Vascular biology of magnesium and its transporters in hypertension.

Magnesium may influence blood pressure by modulating vascular tone and structure through its effects on myriad biochemical reactions that control vascular contraction/dilation, growth/apoptosis, differentiation and inflammation. Magnesium acts as a calcium channel antagonist, it stimulates production of vasodilator prostacyclins and nitric oxide and it alters vascular responses to vasoconstrictor agents. Mammalian cells regulate Mg2+ concentration through special transport systems that have only recently been characterized. Magnesium efflux occurs via Na2+-dependent and Na2+-independent pathways. Mg2+ influx is controlled by recently cloned transporters including Mrs2p, SLC41A1, SLC41A2, ACDP2, MagT1, TRPM6 and TRPM7. Alterations in some of these systems may contribute to hypomagnesemia and intracellular Mg2+ deficiency in hypertension and other cardiovascular pathologies. In particular, increased Mg2+ efflux through dysregulation of the vascular Na+/Mg2+ exchanger and decreased Mg2+ influx due to defective vascular and renal TRPM6/7 expression/activity may be important in altered vasomotor tone and consequently in blood pressure regulation. The present review discusses the role of Mg2+ in vascular biology and implications in hypertension and focuses on the putative transport systems that control magnesium homeostasis in the vascular system. Much research is still needed to clarify the exact mechanisms of cardiovascular Mg2+ regulation and the implications of aberrant cellular Mg2+ transport and altered cation status in the pathogenesis of hypertension and other cardiovascular diseases.

Thyroid-stimulating hormone stimulates lipolysis in adipocytes in culture and raises serum free fatty acid levels in vivo.

Thyroid-stimulating hormone (TSH) stimulates adipocyte lipolysis, but signal transduction pathways activated by TSH for this response have not been directly studied. Using differentiated 3T3-L1 adipocytes as well as primary human adipocytes, we characterized the lipolytic action of TSH with dose-response and time-course studies, and compared it with isoproterenol. Thyroid-stimulating hormone stimulated phosphorylation of perilipin and hormone-sensitive lipase (HSL). Inhibition of protein kinase A with H89 blocked TSH-stimulated lipolysis as well as phosphorylation of perilipin and HSL. Thyroid-stimulating hormone stimulated lipolysis in vivo, as indicated by an elevation in serum free fatty acid (FFA) levels after recombinant human TSH administration to thyroidectomized patients (42% increase, n = 19, P < .05). For patients with a body mass index less than 30 kg/m(2), the TSH-induced increase in serum FFA levels was 53% (n = 11, P < .05), whereas levels in patients with a body mass index of at least 30 kg/m(2) (n = 8) did not change after TSH treatment. In summary, TSH stimulates lipolysis and phosphorylation of perilipin and HSL in a protein kinase A-dependent manner in differentiated adipocytes in culture and raises serum FFA levels in vivo.

Thyroid-stimulating hormone induces interleukin-6 release from human adipocytes through activation of the nuclear factor-kappaB pathway.

Our objective was to identify the signaling pathway activated by TSH that induces IL-6 secretion from human abdominal sc differentiated adipocytes. Human abdominal sc preadipocytes in culture were differentiated into adipocytes. IL-6 release stimulated by TSH was inhibited by 35% (P < 0.05) with SN50, an inhibitor of nuclear factor-kappaB (NF-kappaB) nuclear translocation, and 60% (P < 0.01) with sc-514, an inhibitor of inhibitory-kappaB (IkappaB) kinase (IKK)-beta. Phosphorylation of IKKbeta increased upon TSH treatment (10.3-fold, P < 0.01), and IkappaBalpha levels were reduced by 78% (P < 0.01). TSH activated NF-kappaB (23-fold, P < 0.001), a process that was inhibited (60%, P < 0.01) by SN50. Inhibition of protein kinase A by H89 did not affect TSH-stimulated IKKbeta phosphorylation or IkappaBalpha degradation. TSH-mediated NF-kappaB activation and IL-6 induction also specifically occurred in Chinese hamster ovarian cells expressing the human TSH receptor, resulting in a 5.9-fold (P < 0.001) increase in IKKbeta phosphorylation and a 9.5-fold increase in IL-6 mRNA expression. Our data demonstrate that the IKKbeta/NF-kappaB pathway is a novel TSH target that is required for TSH-induced IL-6 release from human adipocytes.

Thyroid-stimulating hormone stimulates interleukin-6 release from 3T3-L1 adipocytes through a cAMP-protein kinase A pathway.

OBJECTIVE: Thyroid-stimulating hormone (TSH) is a novel modulator of adipokine release from human and mouse adipocytes. The aim of our study was to identify the signal transduction pathways activated by TSH that stimulate interleukin (IL)-6 production. RESEARCH METHODS AND PROCEDURES: Mouse 3T3-L1 preadipocyte and differentiated adipocyte cell cultures were studied. The effect of 0 to 1 microM TSH on IL-6 protein release into the medium over 0 to 24 hours was assessed. TSH signaling pathways responsible for regulating IL-6 were studied through the use of 1 muM forskolin, 100 microM 8-pCPT-2'-O-Me-cAMP, 10 microM H89, 50 microM PD98059, and 2 mug/mL actinomycin D. RESULTS: TSH stimulated IL-6 release by 2.6-fold from 3T3-L1 adipocytes at concentrations as low as 0.01 microM but did not alter IL-6 production of corresponding preadipocytes. Forskolin (elevates intracellular cAMP) stimulated IL-6 release from 3T3-L1 adipocytes (n = 3, p < 0.005), and H89, an inhibitor of cAMP-dependent protein kinase A (PKA), reduced TSH-stimulated IL-6 release by 66% (n = 3, p < 0.01), indicating a requirement for cAMP-dependent PKA. Inhibition of the mitogen-activated protein kinase pathway with PD98059 did not affect TSH-stimulated IL-6 release. Activation of an alternate cAMP target, the exchange protein of cAMP, with 8-pCPT-2'-O-Me-cAMP, had no effect on IL-6 release. TSH raised the level of IL-6 mRNA, and blockade of transcription with actinomycin D abrogated IL-6 protein release by TSH (n = 3, p < 0.05). DISCUSSION: TSH stimulates IL-6 release from differentiated 3T3-L1 adipocytes, but not preadipocytes, by signaling through cAMP-PKA to activate IL-6 gene transcription.