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To determine if ketoacidosis contributes to reduced apolipoprotein A1 (apoA1) expression in insulin-deficient diabetic rats, we examined the regulation of apoA1 gene expression in response to changes in ambient pH or ketone body concentrations. Hepatic apoAI mRNA levels were reduced 42% in diabetic rats relative to nondiabetic controls (means+/-s.d.; 321.8+/-43.7 vs 438.7+/-58.8 arbitrary units; P<0.03). Neither endogenous apoA1 mRNA nor transcriptional activity of the rat apoA1 gene promoter (from -474 to -7) were altered by sodium butyrate or isobutyramide (0.3 mM to 10 mM) in Hep G2 or Caco-2 cells. Rat hepatic and intestinal apoA1 mRNA levels, and plasma apoA1 concentration, were not altered 24 h after isobutyramide administration (500 mg/kg by gavage). When the effect of altering ambient pH within a wide range commonly encountered in vivo was studied, acidosis (pH 6.7), relative to alkalosis (pH 7.9), decreased apoAI mRNA levels relative to glyceraldehyde-3-phosphate dehydrogenase mRNA by 47% in Hep G2 cells (P<0.025) and by 24% in Caco-2 cells (P<0.017). Acidosis did not alter cytomegalo virus (CMV)-beta-galactosidase activity, or the activity of the simian virus (SV40) early-region promoter, in either cell line transfected with the respective constructs. The lowering of ambient pH was associated with a graded reduction in apoAI promoter activity. At pH 6.7, apoAI promoter activity was reduced by 75% compared with promoter activity at pH 7.9. These observations indicate that acidosis, but not ketosis, contributes to the reduction in apoA1 expression during diabetic ketoacidosis by down-regulating apoAI promoter activity.

Stanniocalcin 1 (STC1) is a hypocalcemic hormone that is known to play an important role in calcium metabolism in teleost fish. An increase in blood Ca^{2 +} levels stimulates its synthesis and release. The biological action of STC1 inhibits gill Ca^{2 +} transport (GCAT), but we as yet have no clear understanding of how STC1 inhibits GCAT. In the present study, we characterized the binding, signaling, and action of STC1 on gill cells. Treatment of gill cell cultures with the extracts of corpuscles of Stannius or recombinant STC1 proteins (STC1–V5) led to an increase in cytosolic cAMP levels. Using in situ ligand-binding assays, we demonstrated that STC1–V5 binds to both lamellar and inter-lamellar regions of gill sections. The binding sites were significantly increased in gill sections obtained from fish adapted to high-Ca^{2 +} (2 mM) freshwater (FW) as compared with those from fish adapted to low-Ca^{2 +} (0.2 mM) FW. Receptor-binding assays illustrated specific binding of STC1-alkaline phosphatase to plasma membrane (K _d of 0.36 nM), mitochondria (K _d of 0.41 nM), and nuclear (K _d of 0.71 nM) preparations from gill cells. STC1 binding capacity was significantly greater in the plasma membrane preparations of gills obtained from fish adapted to high-Ca^{2 +} FW. Using isolated pavement cells and mitochondria-rich cells in cAMP assays, we obtained results indicating that both cell types responded to STC1. To illustrate the biological action of STC1, we conducted Ca^{2 +} imaging experiments to demonstrate the effects of STC1 on thapsigargin-induced elevation of cytosolic Ca^{2 +}. Our results indicated that STC1 exerted its inhibitory action via a cAMP pathway to lower intracellular Ca^{2 +} levels. Intriguingly, we were able to block the action of STC1 using an inhibitor, NS-398, of cyclooxygenase-2 (COX-2), which is known to stimulate the activity of sarcoplasmic and endoplasmic reticulum Ca^{2 +}-ATPase (SERCA). A follow-up experiment in which gill cells were incubated with STC1 revealed a downregulation of the epithelial Ca^{2 +} channel (ecacl) but an upregulation of cox-2 expression. The ECaCl is a gatekeeper for Ca^{2 +} entry, whereas COX-2 mediates an activation of SERCA. Taking these results together, the present study is, to our knowledge, the first to provide evidence of STC1 binding and signaling as well as the first to decipher the mechanism of the effect of STC1 on fish gills.

The expression of the Ca^{2 +}-sensing receptor (Casr) in the endocrine gland known as the corpuscle of Stannius (CS) regulates the secretion of the hypocalcemic hormone stanniocalcin-1 (STC1) to inhibit gill Ca^{2 +} uptake. Although numerous studies have reported the branchial expression of Casr and Stc1, the functions of these proteins in gills have not been elucidated yet. On the basis of recent findings regarding the autocrine/paracrine functions of STC1 in mammalian models, we proposed the hypothesis that branchial CaSR has an in situ ‘sensing’ function to regulate STC1 that maintains local Ca^{2 +} homeostasis. In this study, we investigated Casr-mediated signaling and its regulation of Stc1 and cyclooxygenase-2 (Cox2) expression/function using a primary gill-cell culture model. The biochemical responses of gill cells isolated from Japanese eels to an increasing concentration of extracellular Ca^{2 +} (0.1–1 mM) were tested. This stimulation led to a transient increase in phosphatidylcholine-phospholipase C (PC-PLC) activity, followed by activation of ERK and inositol 1,4,5-trisphosphate-Ca^{2 +}/calmodulin-dependent protein kinase 2 (CaMK2) signaling pathways. Cotreatment with the calcimimetic R467 caused synergistic effects on Ca^{2 +}-stimulated PC-PLC activity, ERK signaling, and CaMK2 signaling. The activation of the CaSR-PLC-ERK pathway was associated with increased expression levels of Stc1 and Cox2 as confirmed by the inhibition of Erk using a chemical inhibitor, PD98059. Functionally, Ca^{2 +}/R-467 pretreatment was found to protect cells from thapsigargin-induced cell death. Inhibition of COX2 activity using NS398 abolished this protection, while transduction of STC1 lentiviral particles in the gill cells increased the protective effects. Collectively, our data revealed the expression of functional CaSR in gill tissues. The identification of the CaSR-STC1/COX2-mediated protective pathway in gill cells sheds light on a possible cellular protective mechanism against an increase in intracellular Ca^{2 +} levels associated with transepithelial Ca^{2 +} transport.

Human stanniocalcin 1 (STC1) has recently been identified as a putative protein factor involved in cellular apoptosis. The use of histone deacetylase inhibitor (i.e. trichostatin A (TSA)) and doxorubicin (Dox) is one of the common treatment methods to induce apoptosis in human cancer cells. A study on TSA and Dox-mediated apoptosis may shed light on the regulation and function of STC1 in cancer treatment. In this study, TSA and Dox cotreatment in human nasopharyngeal carcinoma cells (CNE2) elicited synergistic effects on STC1 gene expression and cellular apoptosis. An activation of p53 (TP53) transcriptional activity in Dox- or Dox+TSA-treated cells was revealed by the increased expression levels of p53 mRNA/protein as well as p53-driven luciferase activities. To elucidate the possible involvement of p53 in STC1 gene transcription, a vector expressing wild-type or dominant negative (DN) p53 was transiently transfected into the cells. Both STC1 promoter luciferase constructs and chromatin immunoprecipitation assays did not support the direct role of p53 in STC1 gene transactivation. However, the synergistic effects of p53 on the induction of NF-κB phosphorylation and the recruitment of acetylated histone H3 in STC1 promoter were observed in TSA-cotreated cells. The overexpression of exogenous STC1 sensitized apoptosis in Dox-treated cells. Taken together, this study provides data to show the cross talk of NF-κB, p53, and histone protein in the regulation of STC1 expression and function.

Growth hormone (GH) is known to stimulate luteinizing hormone (LH) release via paracrine interactions between somatotrophs and gonadotrophs. However, it is unclear if LH can exert a reciprocal effect to modulate somatotroph functions. Here we examined the paracrine effects of LH on GH gene expression using grass carp pituitary cells as a cell model. LH receptors were identified in grass carp somatotrophs and their activation by human chorionic gonadotropin (hCG) increased ‘steady-state’ GH mRNA levels. Removal of endogenous LH by immunoneutralization using LH antiserum inhibited GH release and GH mRNA expression. GH secretagogues, including gonadotrophin releasing hormone (GnRH), pituitary adenylate cyclase-activating polypeptide (PACAP) and apomorphine, were effective in elevating GH mRNA levels but these stimulatory actions were blocked by LH antiserum. In pituitary cells pretreated with actinomycin D, the half-life of GH mRNA was not affected by hCG but was enhanced by LH immunoneutralization. Treatment with LH antiserum also suppressed basal levels of mature GH mRNA and primary transcripts. hCG increased cAMP synthesis in carp pituitary cells and hCG-induced GH mRNA expression was mimicked by forskolin but suppressed by inhibiting adenylate cyclase and protein kinase A. Similarly, the stimulatory actions of hCG and forskolin on GH mRNA expression were blocked by inhibiting Janus kinase 2 (JAK2) and MAP kinase (MAPK), including P42/44^MAPK and P38 ^MAPK. These results suggest that LH is essential for the maintenance of GH release, GH gene expression, and somatotroph responsiveness to GH-releasing factors. The paracrine actions of LH on GH mRNA expression are mediated by a concurrent increase in GH gene transcription and GH mRNA turnover, probably through JAK2/MAPK coupled to the cAMP-dependent pathway.

The thymus contains many apoptotic cells that arise from the process of positive and negative selection. Both thymic macrophages and thymic nurse cells/nursing thymic epithelial cells (nursing TECs), non-professional phagocytes, recognize and ingest apoptotic cells without inflammation or tissue damage. Previously we reported that human scavenger receptor class B (SR-B1) is involved in recognition of apoptotic thymocytes by nursing TECs. In this study, we examined the expression and role of a phosphatidylserine receptor (PSR). This receptor is believed to participate in the clearance of apoptotic cells. PSR was strongly expressed in nursing TECs. Transforming growth factor-beta augmented the expression of PSR leading to enhanced binding of apoptotic cells to nursing TECs. In nursing TECs, suppressed expression of human SR-B1 with anti-PSR antibody decreased binding of apoptotic thymocytes to nursing TECs. Our results suggest that both PSR and SR-B1 are expressed in nursing TECs and these receptors appear to play a major role in the clearance of apoptotic cells from the thymus.

Menin is a protein encoded by the gene mutated in multiple endocrine neoplasia type 1 (MEN1) characterized by multiple endocrine tumors of the parathyroid glands, pancreatic islets and the anterior pituitary, especially prolactinoma. In this study, we examined the effects of menin on human prolactin (hPRL) expression. In rat pituitary GH3 cells stably expressing menin, both PRL gene expression/secretion and thymidine incorporation into DNA were inhibited as compared with mock-transfected cells. The transcriptional activity of PRL promoter in GH3 cells co-transfected with menin was significantly decreased. A deletion mutation (569 delC), which we identified in a Japanese MEN1 family, was introduced into menin. When GH3 cells were transfected with a mutant menin expression vector, inhibition of hPRL promoter activity was partially reversed. These observations suggest that menin inhibits hPRL promoter activity and cell proliferation, raising the possibility that menin might play an important role in the tumorigenesis of prolactinoma.