Phosphorylation of a carboxyl-terminal serine within the kappa-opioid receptor produces desensitization and internalization

G-protein receptor kinase and beta-arrestin mediated desensitization of the rat kappa-opioid receptor (KOR) was previously shown using Xenopus oocyte expression to require serine 369 within the C terminus of KOR. To define the effects of phosphorylation of this residue in desensitization and internalization processes in mammalian expression systems, wild-type KOR-green fluorescent protein (KOR-GFP) and KOR(S369A)-GFP were stably expressed in AtT-20 and HEK293 cells. Using whole-cell patch clamp recording in transfected AtT-20 cells, agonist activation of either kappa receptor form produced equivalent activation of the intrinsic G-protein-gated inwardly rectifying potassium channel. Incubation for 60 min with the kappa agonist U50,488 (100 nm) desensitized the response in cells expressing wild-type KOR-GFP by 86% but had no effect on KOR(S369A)-GFP-expressing cells. Phosphorylation of serine 369 was detected using a phosphospecific antibody (KOR-P) able to distinguish the phosphorylated form of the receptor. The agonist-induced increase in KOR-P labeling was dose-dependent, blocked by co-treatment with the kappa antagonist norbinaltorphimine, and prevented by co-expression of the dominant negative form of the G-protein receptor kinase, GRK2(K220R). In contrast, agonist-induced increase in KOR-P labeling was not evident in KOR(S369A) expressing cells. Prolonged activation resulted in receptor internalization that was also blocked by KOR(S369A) substitution, but interestingly, KOR-P labeling was evident at lower agonist concentrations than required to induce internalization. Following the removal of agonist, receptor dephosphorylation detected by loss of KOR-P labeling was complete within 60 min, could be blocked by okadaic acid, and was not blocked by sucrose inhibition of receptor internalization. These results demonstrate that GRK-mediated phosphorylation of serine 369 mediates rat KOR desensitization and internalization.     

Differential contribution of inhibitory phosphorylation of CDC2 and CDK2 for unperturbed cell cycle control and DNA integrity checkpoints

Inhibition of cyclin-dependent kinases (CDKs) by Thr14/Tyr15 phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G2 DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominant-negative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr14/Tyr15 phosphorylation in normal cell cycle and during the DNA damage checkpoint.     

In vitro selection of second site revertants analysis of the hairpin ribozyme active site

We have used in vitro genetics to evaluate the function and interactions of the conserved base G8 in the hairpin ribozyme catalytic RNA. Second site revertant selection for a G8X mutant, where X is any of the other three natural nucleobases, yielded a family of second site suppressors of the G8U mutant, but not of G8C or G8A, indicating that only G and U can be tolerated at position 8 of the ribozyme. This result is consistent with recent observations that point to the functional importance of G8 N-1 in the chemistry of catalysis by this ribozyme reaction. Suppression of the G8U mutation was observed when changes were made directly across loop A from the mutated base at substrate position +2 or positions +2 and +3 in combination. The same changes made in the context of the natural G8 sequence resulted in a very large drop in activity. Thus, the G8U mutation results in a change in specificity of the ribozyme from 5'-N / GUC-3' to 5'-N / GCU-3'. The results presented imply that G8 interacts directly with U+2 during catalysis. We propose that this interaction favors the correct positioning of the catalytic determinants of G8. The implications for the folding of the ribozyme and the catalytic mechanism are discussed.     

Hypoxia inducible factor activates the transforming growth factor-alpha/epidermal growth factor receptor growth stimulatory pathway in VHL(-/-) renal cell carcinoma cells

Bi-allelic-inactivating mutations of the VHL tumor suppressor gene are found in the majority of clear cell renal cell carcinomas (VHL(-/-) RCC). VHL(-/-) RCC cells overproduce hypoxia-inducible genes as a consequence of constitutive, oxygen-independent activation of hypoxia inducible factor (HIF). While HIF activation explains the highly vascularized nature of VHL loss lesions, the relative role of HIF in oncogenesis and loss of growth control remains unknown. Here, we report that HIF plays a central role in promoting unregulated growth of VHL(-/-) RCC cells by activating the transforming growth factor-alpha (TGF-alpha)/epidermal growth factor receptor (EGF-R) pathway. Dominant-negative HIF and enzymatic inhibition of EGF-R were equally efficient at abolishing EGF-R activation and serum-independent growth of VHL(-/-) RCC cells. TGF-alpha is the only known EGF-R ligand that has a VHL-dependent expression profile and its overexpression by VHL(-/-) RCC cells is a direct consequence of HIF activation. In contrast to TGF-alpha, other HIF targets, including vascular endothelial growth factor (VEGF), were unable to stimulate serum-independent growth of VHL(-/-) RCC cells. VHL(-/-) RCC cells expressing reintroduced type 2C mutants of VHL, and which retain the ability to degrade HIF, fail to overproduce TGF-alpha and proliferate in serum-free media. These data link HIF with the overproduction of a bona fide renal cell mitogen leading to activation of a pathway involved in growth of renal cancer cells. Moreover, our results suggest that HIF might be involved in oncogenesis to a much higher extent than previously appreciated.     

Mast cell-mediated apoptosis of endothelial cells in vitro: a paracrine mechanism involving TNF-alpha-mediated down-regulation of bcl-2 expression

Degranulated mast cells are present in the subendothelial space of eroded (de-endothelialized) coronary atheromas. Upon degranulation, mast cells secrete into the surrounding tissue an array of preformed and newly synthesized mediators, including proapoptotic molecules, such as chymase and TNF-alpha. In a co-culture system involving rat serosal mast cells and rat cardiac (microvascular) endothelial cells, we could show, by means of competitive RT-PCR, immunoblotting, immunocytochemistry, annexin staining, flow cytometry, and DNA-laddering, that stimulation of mast cells with ensuing degranulation rapidly (within 30 min) down-regulated the expression of both bcl-2 mRNA and protein, with subsequent induction of apoptosis in the endothelial cells. The major effect of bcl-2 down-regulation resided in the exocytosed granule remnants, a minor effect also being present in the granule remnant-free supernatant. No significant changes were observed in the expression levels of the pro-apoptotic protein, bax. The mast cell-mediated apoptotic effect was partially (70%) dependent on the presence of TNF-alpha and involved the translocation of cytochrome C from mitochondria into cytoplasm. These results are the first to show that one of the cell types present in the atherosclerotic plaques, namely the mast cell, by releasing both granule-remnant-bound and soluble TNF-alpha, may contribute to the erosion of atherosclerotic plaques by inducing apoptosis in adjacent endothelial cells. Published 2003 Wiley-Liss, Inc.     

Contribution of membrane-associated prostaglandin E2 synthase to bone resorption

This study initially confirmed that, among prostaglandins (PGs) produced in bone, only PGE(2) has the potency to stimulate osteoclastogenesis and bone resorption in the mouse coculture system of osteoblasts and bone marrow cells. For the PGE(2) biosynthesis two isoforms of the terminal and specific enzymes, membrane-associated PGE(2) synthase (mPGES) and cytosolic PGES (cPGES) have recently been identified. In cultured mouse primary osteoblasts, both mPGES and cyclooxygenase-2 were induced by the bone resorptive cytokines interleukin-1, tumor necrosis factor-alpha, and fibroblast growth factor-2. Induction of mPGES was also seen in the mouse long bone and bone marrow in vivo by intraperitoneal injection of lipopolysaccharide. In contrast, cPGES was expressed constitutively both in vitro and in vivo without being affected by these stimuli. An antisense oligonucleotide blocking mPGES expression inhibited not only PGE(2) production, but also osteoclastogenesis and bone resorption stimulated by the cytokines, which was reversed by addition of exogenous PGE(2). We therefore conclude that mPGES, which is induced by and mediates the effects of bone resorptive stimuli, may make a target molecule for the treatment of bone resorptive disorders.     

Metabolism of medroxyprogesterone acetate (MPA) via CYP enzymes in vitro and effect of MPA on bleeding time in female rats in dependence on CYP activity in vivo

Medroxyprogesterone acetate (MPA) is a drug commonly used in endocrine therapy for advanced breast cancer, although it is known to cause thrombosis as a serious side effect. Recently, we found that cytochrome P450 3A4 (CYP3A4) mainly catalyzed the metabolism of MPA via CYP in human liver microsomes. However, the metabolic products of MPA in humans and rats have not been elucidated. In addition, it is not clear whether thrombosis could be induced by MPA itself or by its metabolites. In this study, we determined the overall metabolism of MPA as the disappearance of the parent drug from an incubation mixture, and identified the enzymes catalyzing the metabolism of MPA via CYP in rats. Moreover, the effects of CYP-modulators on MPA-induced hypercoagulation in vivo were examined. Intrinsic clearance of MPA in rat liver microsomes was increased by treatment with CYP3A-inducers. The intrinsic clearance of MPA in liver microsomes of rats treated with various CYP-inducers showed a significant correlation with CYP3A activity, but not CYP1A activity, CYP2B activity or CYP2C contents. Among the eight recombinant rat CYPs studied, CYP3A1, CYP3A2 and CYP2A2 catalyzed the metabolism of MPA. However, since CYP3A2 and CYP2A2 are male-specific isoforms, CYP3A1 appears to be mainly involved in the metabolism of MPA in liver microsomes of female rats. In an in vivo study, pretreatment of female rats with SKF525A, an inhibitor of CYPs including CYP3A1, significantly (p < 0.05) enhanced MPA-induced hypercoagulation, whereas pretreatment with phenobarbital, an inducer of CYPs including CYP3A1, reduced it. These findings suggest that CYP-catalyzed metabolism of MPA is mainly catalyzed by CYP3A1 and that MPA-induced hypercoagulation is predominantly caused by MPA itself in female rats.     

Nucleotide changes in the translated region of SCN5A from Japanese patients with Brugada syndrome and control subjects

The mutations of the SCN5A gene have been implicated to play a pathogenetic role in Brugada syndrome, which causes ventricular fibrillation. To determine the Brugada-associated mutations in Japanese patients, facilitate pre-symptomatic diagnosis, and allow genotype-phenotype studies, we screened unrelated patients with Brugada syndrome for mutations. DNAs from 6 Japanese patients were obtained and the sequence in the translated region of SCN5A was determined. We could not find the mutations reported previously, but found 17 sites of nucleotide change, consisting of 7 synonymous and 10 non-synonymous nucleotide changes in our patients. Among them, two non-synonymous nucleotide changes (G1663A and G5227A) are specific to our patients and these changes were not found in 53 healthy controls. In 4 patients out of 6, no specific nucleotide change for Brugada syndrome could be detected. Our findings demonstrating no patient-specific change in the translated region of the SCN5A gene among two thirds of the small number of patients examined here imply that another gene other than the SCN5A may be associated with this disease, supporting previous investigations in Japan and other countries.     

Molecular taxonomy of dermatophytes and related fungi by chitin synthase 1 (CHS1) gene sequences

In the present study, the nucleotide sequences of the CHS1 gene from dermatophytes and related fungi in the genera Chrysosporium, Epidermophyton, Microsporum and Trichophyton were investigated using molecular methods. About 440-bp genomic DNA fragments of the CHS1 gene from 21 species were amplified by polymerase chain reaction (PCR) and sequenced. The CHS1 nucleotide sequences of these fungi showed more than 83% similarity. The molecular taxonomy of the CHS1 gene sequences revealed that Microsporum was genetically distinct from Chrysosporium and Trichophyton, as classified by morphological characteristics. This revised version was published online in June 2006 with corrections to the Cover Date.     

Profound negative regulatory effects by resveratrol on vascular smooth muscle cells: a role of p53-p21(WAF1/CIP1) pathway

We investigated the role of resveratrol, a polyphenol rich in red wine, in cell cycle progression and apoptosis of vascular smooth muscle cells (VSMCs). Resveratrol inhibited the growth of human aortic VSMCs at concentrations as low as 1 microM. This was due to the profound dose-dependent inhibition of DNA synthesis by resveratrol. DNA synthesis was more effectively inhibited when cells were pretreated with resveratrol. Resveratrol caused a dose-dependent increase in intracellular p53 and p21(WAF1/CIP1) levels. At lower concentrations (6.25-12.5 microM), resveratrol effectively blocked cell cycle progression of serum-stimulated VSMCs without inducing apoptosis, while the higher concentration of resveratrol (25 microM) selectively induced apoptosis in the same VSMCs. Intriguingly, however, the same high concentration of resveratrol could not induce apoptosis in quiescent VSMCs. These differential biological effects of resveratrol on quiescent and proliferating VSMCs suggest that resveratrol may be capable of selectively eliminating abnormally proliferating VSMCs of the arterial walls in vivo.