Increased protein kinase A type Iα regulatory subunit expression in parathyroid gland adenomas of patients with primary hyperparathyroidism.

Protein kinase A (PKA) regulatory subunit type Iα (RIα) is a major regulatory subunit that functions as an inhibitor of PKA kinase activity. We have previously demonstrated that elevated RIα expression is associated with diffuse-to-nodular transformation of hyperplasia in parathyroid glands of renal hyperparathyroidism. The aim of the current study was to determine whether or not RIα expression is increased in adenomas of primary hyperparathyroidism (PHPT), because monoclonal proliferation has been demonstrated in both adenomas and nodular hyperplasia. Surgical specimens comprising 22 adenomas and 11 normal glands, obtained from 22 patients with PHPT, were analyzed. Western blot and immunohistochemical analyses were employed to evaluate RIα expression. PKA activities were determined in several adenomas highly expressing RIα. RIα expression was also separately evaluated in chief and oxyphilic cells using the "Allred score" system. Expression of proliferating cell nuclear antigen (PCNA), a proliferation marker, was also immunohistochemically examined. Western blot analysis revealed that 5 out of 8 adenomas highly expressed RIα, compared with normal glands. PKA activity in adenomas was significantly less than in normal glands. Immunohistochemical analysis further demonstrated high expression of RIα in 20 out of 22 adenomas. In adenomas, the greater RIα expression and more PCNA positive cells were observed in both chief and oxyphilic cells. The present study suggested that high RIα expression could contribute to monoclonal proliferation of parathyroid cells by impairing the cAMP/PKA signaling pathway.

Thyroid-hormone-dependent activation of the phosphoinositide 3-kinase/Akt cascade requires Src and enhances neuronal survival.

We have reported previously a non-genomic action of T3 (3,3',5-tri-iodothyronine), which stimulates the PI3K (phosphoinositide 3-kinase)/Akt pathway via p85alpha, the regulatory subunit of PI3K, in human skin fibroblasts. The aim of the present study was to elucidate the mechanism by which T3 activates PI3K, and to investigate the physiological role of this T3 action in neuronal cells. We found that T3 activates PI3K/Akt through Src. First, T3 rapidly induced the activation of Src and Akt in N2a cells expressing TRalpha1 (thyroid hormone receptor alpha1; N2aTRalpha), and both were attenuated by either the addition of a Src inhibitor or Src siRNA. In contrast, a PI3K inhibitor could only block the activation of Akt. Secondly, T3 enhanced TRalpha1-p85alpha-Src complex formation, which was also abrogated by a Src inhibitor. The activation of Src and PI3K/Akt contributes to the anti-apoptotic effect of T3 in N2aTRalpha cells. Moreover, it was also observed in primary cerebral cortical neurons that T3 induced the activation of PI3K/Akt and suppressed serum-deprivation-induced apoptosis. Together, the findings of the present study demonstrate a novel non-genomic action of T3 on neuronal cell survival, and provide new insights into the mechanism underlying this action, which involves Src activation and TRalpha1-p85alpha-Src complex formation.

Thyroid hormone non-genomically suppresses Src thereby stimulating osteocalcin expression in primary mouse calvarial osteoblasts.

To provide further insights into non-genomic action of thyroid hormone (T3), we investigated whether Src is under control of T3 in primary calvarial osteoblasts prepared from neonatal mice. Treatment of the cells with T3 rapidly decreased Src Y416 autophosphorylation, followed by the decrease of phosphorylated extracellular signal-regulated kinases, suggesting that T3 non-genomically suppresses Src activity. Furthermore, this T3 effect was rapid and persistent, and was associated with the increased expression of osteocalcin (OC). To confirm the contribution of Src to the effect of T3 on OC expression, a constitutively active Src (Y527F) was overexpressed in osteoblasts. In such cells, Y416 phosphorylation was markedly increased even in the presence of T3, and T3-dependent expression of OC was markedly attenuated. The present study demonstrates a novel, non-genomic action of T3 in primary mouse osteoblasts, by which T3 suppresses Src thereby stimulating OC expression.

Prostaglandin E2 negatively regulates AMP-activated protein kinase via protein kinase A signaling pathway.

We investigated possible involvement of prostaglandin (PG) E2 in regulation of AMP-activated protein kinase (AMPK). When osteoblastic MG63 cells were cultured in serum-deprived media, Thr-172 phosphorylation of AMPK alpha-subunit was markedly increased. Treatment of the cells with PGE2 significantly reduced the phosphorylation. Ser-79 phosphorylation of acetyl-CoA carboxylase, a direct target for AMPK, was also reduced by PGE2. On the other hand, PGE2 reciprocally increased Ser-485 phosphorylation of the alpha-subunit that could be associated with inhibition of AMPK activity. These effects of PGE2 were mimicked by PGE2 receptor EP2 and EP4 agonists and forskolin, but not by EP1 and EP3 agonists, and the effects were suppressed by an adenylate cyclase inhibitor SQ22536 and a protein kinase A inhibitor H89. Additionally, the PGE2 effects were duplicated in primary calvarial osteoblasts. Together, the present study demonstrates that PGE2 negatively regulates AMPK activity via activation of protein kinase A signaling pathway.

3beta-Hydroxysteroid-delta24 reductase is a hydrogen peroxide scavenger, protecting cells from oxidative stress-induced apoptosis.

3beta-Hydroxysteroid-Delta24 reductase (DHCR24) is an endoplasmic reticulum-resident, multifunctional enzyme that possesses antiapoptotic and cholesterol-synthesizing activities. To clarify the molecular basis of the former activity, we investigated the effects of hydrogen peroxide (H(2)O(2)) on embryonic fibroblasts prepared from DHCR24-knockout mice (DHCR24(-/-) mouse embryonic fibroblasts). H(2)O(2) exposure rapidly induced apoptosis, which was associated with sustained activation of apoptosis signal-regulating kinase-1 and stress-activated protein kinases, such as p38 MAPK and c-Jun N-terminal kinase. Complementation of the mouse embryonic fibroblasts by adenovirus expressing DHCR24 attenuated the H(2)O(2)-induced kinase activation and apoptosis. Concomitantly, intracellular generation of reactive oxygen species (ROS) in response to H(2)O(2) was also diminished by the adenovirus, suggesting a ROS-scavenging activity of DHCR24. Such antiapoptotic effects of DHCR24 were duplicated in pheochromocytoma PC12 cells infected with adenovirus. In addition, it was found that DHCR24 exerted cytoprotective effects in the tunicamycin-induced endoplasmic reticulum stress by eliminating ROS. Finally, using in vitro-synthesized and purified proteins, DHCR24 and its C-terminal deletion mutant were found to exhibit high H(2)O(2)-scavenging activity, whereas the N-terminal deletion mutant lost such activity. These results demonstrate that DHCR24 can directly scavenge H(2)O(2), thereby protecting cells from oxidative stress-induced apoptosis.

Thyroid hormone activates adenosine 5'-monophosphate-activated protein kinase via intracellular calcium mobilization and activation of calcium/calmodulin-dependent protein kinase kinase-beta.

AMP-activated protein kinase (AMPK) is a key regulator of glucose and fatty acid homeostasis. In muscle cells, AMPK stimulates mitochondrial fatty acid oxidation and ATP production. The thyroid hormone T3 increases cellular oxygen consumption and is considered to be a major regulator of mitochondrial activities. In this study, we examined the possible involvement of AMPK in the stimulatory action of T3 on mitochondria. Treatment of C2C12 myoblasts with T3 rapidly led to phosphorylation of AMPK. Acetyl-coenzyme A carboxylase, a direct target of AMPK, was also phosphorylated after T3 treatment. Similar results were obtained with 3T3-L1, FRTL-5, and HeLa cells. Stable expression of T3 receptor (TR)-alpha or TRbeta in Neuro2a cells enhanced this effect of T3, indicating the involvement of TRs. Because HeLa cells express only Ca2+/calmodulin-dependent protein kinase kinase-beta (CaMKKbeta), one of two known AMPK kinases, it was suggested that the effect of T3 is mediated by CaMKKbeta. Indeed, experiments using a CaMKK inhibitor, STO-609, and an isoform-specific small interfering RNA demonstrated the CaMKKbeta-dependent phosphorylation of AMPK. Furthermore, T3 was found to rapidly induce intracellular Ca2+ mobilization in HeLa cells, and a Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), suppressed T3- as well as ionomycin-dependent phosphorylation of AMPK. In addition, T3-dependent oxidation of palmitic acids was attenuated by BAPTA, STO-609, and the small interfering RNA for CaMKKbeta, indicating that T3-induced activation of AMPK leads to increased fatty acid oxidation. These results demonstrate that T3 nontranscriptionally activates AMPK via intracellular Ca2+ mobilization and CaMKKbeta activation, thereby stimulating mitochondrial fatty acid oxidation.

Long-term amiodarone treatment causes cardioselective hypothyroid-like alteration in gene expression profile.

The long-term cardiac effects of amiodarone resemble many aspects of hypothyroidism. The anti-arrhythmic potential of amiodarone may therefore be the result of a drug-induced, local hypothyroid-like condition. To investigate this controversial issue, we compared gene expression profiles in the hearts of rats treated with amiodarone with those of rats with hypothyroidism. Wistar male rats were assigned to 3 groups (n=6-8): Control, systemic hypothyroidism (hypothyroidism) and amiodarone treatment (amiodarone, 150 mg/kg/day, p.o., 4 weeks). Electrocardiogram (ECG) recordings, gene profiling by DNA microarray and Northern blotting were carried out. Amiodarone, like hypothyroidism, caused significant prolongation of RR and QT intervals in ECGs. Microarray analysis of 8435 genes in the left ventricular myocardium revealed a significant similarity in expression profiles between hypothyroidism and amiodarone (R=0.63, p<0.00001). The gene expression profiles of hypothyroidism and amiodarone showed closer correlation when top 100 up-regulated and 100 down-regulated genes in hypothyroidism (total 200 genes) were analyzed (R=0.78, p<0.00001). Northern blots of left ventricular myocardium showed a parallel decrease in mRNAs for myosin heavy chain (MHC)-alpha and a parallel increase for myosin heavy chain (MHC)-beta in hypothyroidism and amiodarone. In the liver and pituitary, in contrast, Northern blots showed quite different changes in the transcripts of the representative T3-responsive genes in the hypothyroidism and amiodarone. In conclusion, long-term treatment with amiodarone causes cardioselective hypothyroid-like alterations in gene expression profiles. The potent anti-arrhythmic activity of amiodarone may be attributable, in part at least, to this unique transcriptional remodeling.

Insulin-like growth factor-I activation of Akt survival cascade in neuronal cells requires the presence of its cognate receptor in caveolae.

Insulin-like growth factor-I (IGF-I) plays important roles in survival of neurons. Caveolae, cholesterol-rich microdomains of plasma membrane, act as platforms for some neurotrophic factors. In this study, we examined a possible role of caveolae in IGF-I signal transduction in pheochromocytoma PC12 cells. IGF-I treatment attenuated serum withdrawal-induced apoptosis, which was reversed by treatment with methyl-beta-cyclodextrin (CD) that removes cholesterol from plasma membrane. Immunocytochemical and subcellular fractionation analyses revealed that IGF-I receptor (IGF-IR) was colocalized with caveolin-1, a major protein component in caveolae, and that CD treatment reduced IGF-IR contents in caveolae. Consistent with these findings, IGF-I phosphorylation of insulin receptor substrate-1 and Akt was impaired, and cholesterol supply restored the IGF-I action. Furthermore, experiments using small interfering RNA revealed that the reduction of caveolin-1 expression impaired the IGF-I action. In addition, the colocalization of IGF-IR with caveolin-1, and the caveolae-dependent IGF-I action were duplicated in primary culture of rat cerebellar granule neurons. These results demonstrate that the presence of IGF-IR in caveolae is required for the neuroprotective action of IGF-I.

Parathyroid hormone activates phosphoinositide 3-kinase-Akt-Bad cascade in osteoblast-like cells.

To understand the molecular basis underlying the anabolic action of parathyroid hormone (PTH) on bone, the anti-apoptotic action of PTH on osteoblast-like cells was investigated. Since Akt is a key protein kinase for cell survival, we focused on a possible involvement of Akt in the anti-apoptotic action of PTH. Human osteoblast-like MG-63 cells cultured without serum were treated with PTH. Western blot analysis revealed that PTH rapidly phosphorylated Akt and induced its nuclear translocation. The phosphorylation of pro-apoptotic protein Bad was also increased by PTH, leading to its inactivation. The PTH-dependent activation of Akt was also detected in other osteoblastic cell lines, SaOS-2 and ROS 17/2.8. The pretreatment of MG-63 cells with either one of inhibitors for phosphoinositide 3-kinase (PI3K), wortmannin or LY294002 prevented Akt and Bad phosphorylation. Furthermore, co-immunoprecipitation analysis revealed that PTH receptor (PTH-1R) directly interacted with p85, a regulatory subunit of PI3K, in a PTH-dependent manner. Serum withdrawal induced the apoptosis of MG-63 cells, and PTH prevented the apoptosis, which was inhibited by PI3K inhibitors. These results demonstrate the presence of a novel PTH/PTH receptor signaling cascade consisting of PTH-1R, PI3K, Akt and Bad and that this cascade can work as an anti-apoptotic signaling pathway in osteoblast-like cells.

Molecular cloning of prostaglandin EP3 receptors from canine sensory ganglia and their facilitatory action on bradykinin-induced mobilization of intracellular calcium.

We previously demonstrated that the activation of prostaglandin E-prostanoid-3 (EP3) receptor sensitized the canine nociceptor response to bradykinin (BK). To elucidate the molecular mechanism for this sensitization, we cloned two cDNAs encoding EP3s with different C-terminals, from canine dorsal root ganglia, and established the transformed cell lines stably expressing them. In both transformants, EP3 agonist did not increase intracellular cAMP levels, but it attenuated forskolin-dependent cAMP accumulation in a pertussis toxin (PTX)-sensitive manner and increased intracellular calcium levels in a PTX-resistant manner, indicating that both EP3s can couple with Gi and Gq, but not with Gs proteins. As the nociceptor response to BK is mediated by BK B2 receptor, it was transfected into the transformants and the effects of EP3 agonist on BK-dependent calcium mobilization were investigated. When BK was applied twice with a 6-min interval, the second response was markedly attenuated. Pre-treatment with EP3 agonist had no effect on the initial response, but restored the second response in a PTX-sensitive manner. A protein kinase A inhibitor mimicked the effect of EP3 agonist. These results demonstrate that the activation of EP3 restores the response to BK by attenuating the desensitization of BK B2 receptor activity via Gi protein.

Differential expression of cyclin-dependent kinase inhibitors, p27Kip1 and p57Kip2, by corticotropin in rat adrenal cortex.

An important role for the cyclin-dependent kinase inhibitors (CDKIs), p27Kip1 and p57Kip2, in the proliferation and differentiation of adrenal cells has been suggested by their knockout mice, which display adrenal hyperplasia. Adrenal development and function are primarily regulated by ACTH. In the present study, we investigated the effects of ACTH on the expression of p27Kip1, p57Kip2 and proliferating cell nuclear antigen (PCNA) in rat adrenals. Male Wistar rats were treated with dexamethasone (Dex) to inhibit endogenous ACTH secretion. ACTH was then administered to the rats, and the adrenals were examined by Western blot and immunohistochemical analyses. Dex treatment induced shrinkage of adrenals where no PCNA-expressing cells were detected, but most of the cells expressed p27Kip1. Subsequent ACTH treatment resulted in the marked suppression of p27Kip1 expression, specifically in adrenocortical cells at 12 h after the stimulus. At 48 h, the p27Kip1 suppression still continued in the cortex, while the PCNA-expressing cells appeared mainly around the zona glomerulosa and increased at 72 h. At this time, the p27Kip1-expressing cells also appeared in the same zone. In contrast to p27Kip1, the expression of p57Kip2 was not detected in the Dex-treated adrenal. However, its expression was markedly induced by ACTH in the zona glomerulosa at 48 and 72 h. The results demonstrate that the primary site for mitogenic action of ACTH in rat adrenocortex is the zona glomerulosa, and that ACTH modulates proliferation of adrenocortical cells by regulating p27Kip1 and p57Kip2 expression in a time- and site-specific manner.

DHCR24-knockout embryonic fibroblasts are susceptible to serum withdrawal-induced apoptosis because of dysfunction of caveolae and insulin-Akt-Bad signaling.

The DHCR24 gene encodes an enzyme catalyzing the last step of cholesterol biosynthesis, the conversion of desmosterol to cholesterol. To elucidate the physiological significance of cholesterol biosynthesis in mammalian cells, we investigated proliferation of mouse embryonic fibroblasts (MEFs) prepared from DHCR24(-/-) mice. Both DHCR24(-/-) and wild-type MEFs proliferated in the presence of serum in culture media. However, the inhibition of external cholesterol supply by serum withdrawal induced apoptosis of DHCR24(-/-) MEFs, which was associated with a marked decrease in the intracellular and plasma membrane cholesterol levels, Akt inactivation, and Bad dephosphorylation. Insulin is an antiapoptotic factor capable of stimulating the Akt-Bad cascade, and its receptor (IR) is enriched in caveolae, cholesterol-rich microdomains of plasma membrane. We thus analyzed the association of IR and caveolae in the cholesterol-depleted MEFs. Subcellular fractionation and immunocytochemical analyses revealed that the IR and caveolin-1 contents were markedly reduced in the caveolae fraction of the MEFs, suggesting the disruption of caveolae, and that large amounts of IR were present apart from caveolin-1 on plasma membrane, indicating the uncoupling of IR with caveolae. Consistent with these findings, insulin-dependent phosphorylations of insulin receptor substrate-1, Akt, and Bad were impaired in the cholesterol-depleted MEFs. However, this impairment was partial because treatment of the MEFs with insulin restored Akt activation and prevented apoptosis. Cholesterol supply also prevented apoptosis. These results demonstrate that the cellular cholesterol biosynthesis is critical for the activation and maintenance of the Akt-Bad cell survival cascade in response to growth factors such as insulin.

DHCR24 gene knockout mice demonstrate lethal dermopathy with differentiation and maturation defects in the epidermis.

Desmosterolosis is an autosomal recessive disorder due to mutations in the 3beta-hydroxysterol-Delta24 reductase (DHCR24) gene that encodes an enzyme catalyzing the conversion of desmosterol to cholesterol. To date, only two patients have been reported with severe developmental defects including craniofacial abnormalities and limb malformations. We employed mice with targeted disruption of DHCR24 to understand the pathophysiology of desmosterolosis. All DHCR24-/- mice died within a few hours after birth. Their skin was wrinkleless and less pliant, leading to restricted movement and inability to suck (empty stomach). DHCR24 gene was expressed abundantly in the epidermis of control but not of DHCR24-/- mice. Accordingly, cholesterol was not detected whereas desmosterol was abundant in the epidermis of DHCR24-/- mice. Skin histology revealed thickened epidermis with few and smaller keratohyaline granules. Aberrant expression of keratins such as keratins 6 and 14 suggested hyperproliferative hyperkeratosis with undifferentiated keratinocytes throughout the epidermis. Altered expression of filaggrin, loricrin, and involcrin were also observed in the epidermis of DHCR24-/-. These findings suggested impaired skin barrier function. Indeed, increased trans-epidermal water loss and permeability of Lucifer yellow were observed in DHCR24-/- mice. DHCR24 thus plays crucial role for skin development and its proper function.

Fenofibrate activates AMPK and increases eNOS phosphorylation in HUVEC.

Fenofibrate improves endothelial function by lipid-lowering and anti-inflammatory effects. Additionally, fenofibrate has been demonstrated to upregulate endothelial nitric oxide synthase (eNOS). AMP-activated protein kinase (AMPK) has been reported to phosphorylate eNOS at Ser-1177 and stimulate vascular endothelium-derived nitric oxide (NO) production. We report here that fenofibrate activates AMPK and increases eNOS phosphorylation and NO production in human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with fenofibrate increased the phosphorylation of AMPK and acetyl-CoA carboxylase. Fenofibrate simultaneously increased eNOS phosphorylation and NO production. Inhibitors of protein kinase A and phosphatidylinositol 3-kinase failed to suppress the fenofibrate-induced eNOS phosphorylation. Neither bezafibrate nor WY-14643 activated AMPK in HUVEC. Furthermore, fenofibrate activated AMPK without requiring any transcriptional activities. These results indicate that fenofibrate stimulates eNOS phosphorylation and NO production through AMPK activation, which is suggested to be a novel characteristic of this agonist and unrelated to its effects on peroxisome proliferator-activated receptor alpha.

Up-regulation of the gene encoding protein kinase A type I alpha regulatory subunit in nodular hyperplasia of parathyroid glands in patients with chronic renal failure.

CONTEXT: Hyperplasia of parathyroid glands in patients with chronic renal failure is classified into diffuse (DH) and nodular (NH) types, and NH is often refractory to routine medical therapy. OBJECTIVE: Although it is considered that the parenchymal cells initially proliferate diffusely and then some of them are transformed to form nodules consisting of monoclonal cells, the underlying molecular mechanism for such a transformation is not fully understood. In this study we tried to identify the genes that are up-regulated in NH. DESIGN AND SETTING: The cDNA population prepared from DH was subtracted from that prepared from NH by a PCR-based cDNA subtraction method. The resultant cDNAs were cloned and sequenced. To confirm the up-regulation of the identified genes, a total of 35 parathyroid glands (18 DH, 16 NH, and one mixed) obtained from 21 patients were analyzed. RESULTS: One of the nuclear genes identified was the PRKAR1A gene, which encodes type Ialpha regulatory subunit (RIalpha) of cAMP-dependent protein kinase (PKA). Immunohistochemical analysis demonstrated that RIalpha was abundantly expressed in the nodular region, whereas the adjacent diffuse region displayed relatively low expression. Northern and Western blot analyses demonstrated up-regulation of RIalpha expression in most NH tested. Determination of PKA activities revealed that free PKA activities measured in the absence of cAMP in the assay were inversely correlated with RIalpha expression, indicating the functional significance of RIalpha up-regulation. CONCLUSIONS: These results suggest that the aberrant expression of RIalpha is involved in the diffuse to nodular transformation of hyperplasia of parathyroid glands by impairing cAMP/PKA signal transduction.

Glutathionylation of two cysteine residues in paired domain regulates DNA binding activity of Pax-8.

We reported that the first two cysteine residues out of three present in paired domain (PD), a DNA-binding domain, are responsible for redox regulation of Pax-8 DNA binding activity. We show that glutathionylation of these cysteines has a regulatory role in PD binding. Wild-type PD and its mutants with substitution of cysteine to serine were synthesized and named CCC, CSS, SCS, SSC, and SSS according to the positions of substituted cysteines. They were incubated in a buffer containing various ratios of GSH/GSSG and subjected to gel shift assay. Binding of CCC, CSS, and SCS was impaired with decreasing GSH/GSSG ratio, whereas that of SSC and SSS was not affected. Because [3H]glutathione was incorporated into CCC, CSS, and SCS, but not into SSC and SSS, the binding impairment was ascribed to glutathionylation of the redox-reactive cysteines. This oxidative inactivation of PD binding was reversed by a reductant dithiothreitol and by redox factor (Ref)-1 in vitro. To explore the glutathionylation in cells, Chinese hamster ovary cells overexpressing CSS and SCS were labeled with [35S]cysteine in the presence of cycloheximide. Immunoprecipitation with an antibody against PD revealed that treatment of the cells with an oxidant diamide induced the 35S incorporation into both mutants, suggesting the PD glutathionylation in cells. Since the two cysteine residues in PD are conserved in all Pax members, this novel posttranslational modification of PD would provide a new insight into molecular basis for modulation of Pax function.

Requirement of thyrotropin-dependent complex formation of protein kinase A catalytic subunit with inhibitor of {kappa}B proteins for activation of p65 nuclear factor-{kappa}B by tumor necrosis factor-{alpha}.

We previously demonstrated that TNF-alpha-dependent activation of p65 nuclear factor kappaB in rat thyroid FRTL-5 cells requires TSH. In the present study, we investigated the mechanism of this TSH action. Western blot analysis revealed that, in both the presence and absence of TSH, degradation of a cytosolic kappaB inhibitor (IkappaBalpha) occurred in response to TNF-alpha, resulting in nuclear translocation of p65 in both conditions. However, no DNA binding of p65 was detected in the absence of TSH, suggesting that posttranslational modification of p65 by TSH is required for its binding. Treatment of the cells cultured in the presence of TSH with a protein kinase A (PKA) inhibitor, H89, markedly reduced p65 binding and its transcriptional activity. However, transient block of TSH/cAMP-dependent activation of PKA catalytic subunit (PKAc) by adenylate cyclase inhibitor, SQ22536, had no effects on the p65 activation. Interestingly, it was found that PKAc formed a complex with IkappaBalpha and beta only in the presence of TSH, and this PKAc could be activated by TNF-alpha. TNF-alpha-dependent p65 activation was temporally associated with PKAc/IkappaBalpha complex formation. More than 3 h exposure of TSH was required for the complex formation and p65 activation. These results demonstrate that TSH induces the formation of PKAc/IkappaB complex in FRTL-5 cells and that this PKAc bound with IkappaB plays a critical role in TNF-alpha-dependent activation of p65.

Thyroid hormone induces rapid activation of Akt/protein kinase B-mammalian target of rapamycin-p70S6K cascade through phosphatidylinositol 3-kinase in human fibroblasts.

We have demonstrated that T3 increases the expression of ZAKI-4alpha, an endogenous calcineurin inhibitor. In this study we characterized a T3-dependent signaling cascade leading to ZAKI-4alpha expression in human skin fibroblasts. We found that T3-dependent increase in ZAKI-4alpha was greatly attenuated by rapamycin, a specific inhibitor of a protein kinase, mammalian target of rapamycin (mTOR), suggesting the requirement of mTOR activation by T3. Indeed, T3 activated mTOR rapidly through S2448 phosphorylation, leading to the phosphorylation of p70(S6K), a substrate of mTOR. This mTOR activation is mediated through phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) signaling cascade because T3 induced Akt/PKB phosphorylation more rapidly than that of mTOR, and these T3-dependent phosphorylations were blocked by both PI3K inhibitors and by expression of a dominant negative PI3K (Deltap85alpha). Furthermore, the association between thyroid hormone receptor beta1 (TRbeta1) and PI3K-regulatory subunit p85alpha, and the inhibition of T3-induced PI3K activation and mTOR phosphorylation by a dominant negative TR (G345R) demonstrated the involvement of TR in this T3 action. The liganded TR induces the activation of PI3K and Akt/PKB, leading to the nuclear translocation of the latter, which subsequently phosphorylates nuclear mTOR. The rapid activation of PI3K-Akt/PKB-mTOR-p70(S6K) cascade by T3 provides a new molecular mechanism for thyroid hormone action.