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.

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.

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.

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.

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.

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.

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.