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Biochemical Pharmacology (v.83, #5)

Editorial Board (pp. iii).

Peptide therapeutics for CNS indications by Paul McGonigle (pp. 559-566).

Neuropeptides play a crucial role in the normal function of the central nervous system and peptide receptors hold great promise as therapeutic targets for the treatment of several CNS disorders. In general, the development of peptide therapeutics has been limited by the lack of drug-like properties of peptides and this has made it very difficult to transform them into marketable therapeutic molecules. Some of these challenges include poor in vivo stability, poor solubility, incompatibility with oral administration, shelf stability, cost of manufacture. Recent technical advances have overcome many of these limitations and have led to rapid growth in the development of peptides for a wide range of therapeutic indications such as diabetes, cancer and pain. This review examines the therapeutic potential of peptide agonists for the treatment of major CNS disorders such as schizophrenia, anxiety, depression and autism. Both clinical and preclinical data has been accumulated supporting the potential utility of agonists at central neurotensin, cholecystokinin, neuropeptide Y and oxytocin receptors. Some of the successful approaches that have been developed to increase the stability and longevity of peptides in vivo and improve their delivery are also described and potential strategies for overcoming the major challenge that is unique to CNS therapeutics, penetration of the blood–brain barrier, are discussed.

Keywords: Peptides; CNS indications; Peptide therapeutics; Oxytocin; Amylin

Pre-exposure chemoprophylaxis of HIV infection: Quo vadis? by Erik De Clercq (pp. 567-573).

Tenofovir is essential part of pre-exposure chemoprophylaxis (PrEP) of HIV infection.The pre-exposure chemoprophylaxis (now commonly referred to as PrEP) of HIV infection has gained increased momentum, concomitantly with the successful use of combination drug regimens for the treatment of AIDS. A pivotal component in the current drug combination regimens for the treatment of AIDS as well as the ongoing PrEP trials is tenofovir disoproxil fumarate (TDF, Viread^®) and its combination with emtricitabine (FTC). The combination of TDF with FTC has been marketed as Truvada^®. TDF and TDF/FTC has proven effective, if orally administered daily or intermittently, in the prevention of rectal simian human immunodeficiency virus (SHIV) infection in macaques. Topical tenofovir gel has proven effective in the prevention of HIV infection in women in South Africa. Oral TDF/FTC has proven effective in the prevention of HIV infection in men having sex with men, and recent press releases divulged that oral TDF/FTC is also effective in preventing HIV infection in serodiscordant couples in Botswana, Kenya and Uganda. Other PrEP studies are still ongoing. Available data point to the efficacy and safety of TDF with or without FTC in the prophylaxis of HIV infection (AIDS).

Keywords: PrEP (pre-exposure prophylaxis); TDF (tenofovir disoproxil fumarate); FTC (emtricitabine); Prophylaxis; HIV; AIDS

Homologous recombination repair-dependent cytotoxicity of the benzotriazine di- N-oxide CEN-209: Comparison with other hypoxia-activated prodrugs by Francis W. Hunter; Jingli Wang; Rita Patel; Huai-Ling Hsu; Anthony J.R. Hickey; Michael P. Hay; William R. Wilson (pp. 574-585).

CEN-209 (SN30000) is a second-generation benzotriazine di- N-oxide currently in advanced preclinical development as a hypoxia-activated prodrug (HAP). Herein we describe the DNA repair-, hypoxia- and one-electron reductase-dependence of CEN-209 cytotoxicity. We deployed mutant CHO cell lines to generate DNA repair profiles for CEN-209, and compared the profiles with those for other HAPs. Hypoxic selectivity of CEN-209 was significantly greater than PR-104A and the nitro-chloromethylbenzindoline (nCBI/SN29428) and comparable to tirapazamine and TH-302. CEN-209 was selective for homologous recombination (HR) repair-deficient cells ( Rad51d^−/−), but less so than nitrogen mustard prodrugs TH-302 and PR-104A. Further, DNA repair profiles for CEN-209 differed under oxic and hypoxic conditions, with oxic cytotoxicity more dependent on HR. This feature was conserved across all three members of the benzotriazine di- N-oxide class examined (tirapazamine, CEN-209 and CEN-309/SN29751). Enhancing one-electron reduction of CEN-209 by forced expression of a soluble form of NADPH:cytochrome P450 oxidoreductase (sPOR) increased CEN-209 cytotoxicity more markedly under oxic than hypoxic conditions. Comparison of oxygen consumption, H₂O₂ production and metabolism of CEN-209 to the corresponding 1-oxide and nor-oxide reduced metabolites suggested that enhanced oxic cytotoxicity in cells with high one-electron reductase activity is due to futile redox cycling. This study supports the hypothesis that both oxic and hypoxic cell killing by CEN-209 is mechanistically analogous to tirapazamine and is dependent on oxidative DNA damage repaired via multiple pathways. However, HAPs that generate DNA interstrand cross-links, such as TH-302 and PR-104, may be more suitable than benzotriazine di- N-oxides for exploiting reported HR repair defects in hypoxic tumour cells.

Keywords: Hypoxia-activated prodrug; Homologous recombination repair; Redox cycling; CEN-209; Tirapazamine; TH-302

Apatinib (YN968D1) enhances the efficacy of conventional chemotherapeutical drugs in side population cells and ABCB1-overexpressing leukemia cells by Xiu-zhen Tong; Fang Wang; Shu Liang; Xu Zhang; Jie-hua He; Xing-Gui Chen; Yong-ju Liang; Yan-jun Mi; Kenneth Kin Wah To; Li-wu Fu (pp. 586-597).

Apatinib increases the intracellular accumulation of anti-cancer drugs (D) that are substrates of ABCB1 and ABCG2 via directly inhibiting the drug transport function.P-glycoprotein (P-gp, ABCB1) overexpression and enrichment of stem-like cells are linked to poor prognosis in tumor patients. In this study, we investigated the effect of apatinib, an oral multi-targeted tyrosine kinase inhibitor (TKI) on enhancing the efficacy of conventional anticancer drugs in side population (SP) cells and ABCB1-overexpressing leukemia cells in vitro, in vivo and ex vivo. Our results showed that apatinib significantly enhanced the cytotoxicity and cell apoptosis induced by doxorubicin in SP cells sorted from K562 cells. Furthermore, apatinib also strongly reversed multidrug resistance (MDR) in K562/ADR cells, and the primary leukemia blasts overexpressing ABCB1 while showed no synergistic interactions with chemotherapeutic agents in MRP1-, MRP4-, MRP7- and LRP-overexpressing cells. Apatinib treatment markedly increased the intracellular accumulation of doxorubicin and rhodamine 123 in K562/ADR cells and the accumulation of rhodamine 123 in the primary leukemia blasts with ABCB1 overexpression. Apatinib stimulated the ATPase activity of P-gp in a dose-dependent manner but did not alter the expression of ABCB1 at both mRNA and protein levels. The phosphorylation level of AKT and ERK1/2 remained unchanged after apatinib treatment in both sensitive and MDR cells. Importantly, apatinib significantly enhanced the antitumor activity of doxorubicin in nude mice bearing K562/ADR xenografts. Taken together, our results suggest that apatinib could target to SP cells and ABCB1-overexpressing leukemia cells to enhance the efficacy of chemotherapeutic drugs. These findings should be useful for the combination of apatinib and chemotherapeutic agents in the clinic.

Keywords: Multidrug resistance; ATP-binding cassette transporter; P-glycoprotein; Leukemia stem-like cells; Xenograft

An indirubin derivative, E804, exhibits potent angiosuppressive activity by Yuk-Kit Chan; Hoi-Hin Kwok; Lai-Sheung Chan; Kelvin Sze-Yin Leung; Jue Shi; Nai-Ki Mak; Ricky Ngok-Shun Wong; Patrick Ying-Kit Yue (pp. 598-607).

Angiogenesis, the development of neovessels from pre-existing vessels, is obligatory for solid tumors survival, growth, invasion, and metastasis. Many anti-angiogenic agents are small molecules originated from natural sources. Recently, angiosuppressive effects of indirubin and its derivatives, the active components in indigo-producing herbs, have been shown to possess anti-viral and anti-inflammatory potentials. In this study, we identified another indirubin derivative, indirubin-3′-(2,3 dihydroxypropyl)-oximether (E804), could exhibit potent angiosuppressive effects. In vitro study showed that E804 could significantly inhibit human umbilical vein endothelial cells proliferation, migration, and tube formation in a concentration-dependent manner (0.4–40μM); at the concentration of 1μmol or above, angiosuppressive potency of E804 was found to be more significant than indirubin-3′-oxime. Using in vivo Matrigel plug model and directed- in vivo-angiogenesis-assay (DIVAA), E804 was shown more effective to attenuate the VEGF/bFGF-induced neovessel formation. The hemoglobin content and the invaded endothelial cells in the implants were also greatly reduced. Results from the aortic ring assay indicated E804 (4μM) could completely suppress ex vivo sprouting of endothelial cells from the rat aorta fragments; with concomitant reduction of gelatinolytic activities of matrix metalloproteinase-2 and -9. E804 also concentration-dependently (0.04–10μM) inhibited the subintestinal vessels formation in zebrafish embryos. This study provides the first evidence that E804, a novel indirubin derivative, could more effectively inhibit angiogenesis. With the improved anti-angiogenic potency when compared with indirubin-3′oxime, E804 would be a new potential candidate in the treatment of angiogenesis-dependent diseases.

Keywords: Anti-angiogenesis; Indirubin-3′-oxime; E804; HUVEC

Urocontrin, a novel UT receptor ligand with a unique pharmacological profile by David Chatenet; Quang-Trinh Nguyen; Myriam Létourneau; Jocelyn Dupuis; Alain Fournier (pp. 608-615).

Urocontrin is able to block both in vivo and in vitro hUII-associated actions but not URP-mediated effects. This new derivative represents the first compound of a new class of urotensinergic ligands.In recent years, several studies have demonstrated that urotensin II (UII) and urotensin II-related peptide (URP) can exhibit differential biological activity. So far, known antagonists of the urotensin II receptor (UT) are of limited usefulness for investigating the specific pathophysiological role of UII or URP. Therefore, identification of new compounds able to discriminate UII- and URP-associated biological activities is crucially needed. In the present study, we report preliminary data regarding the pharmacological properties of a novel UT ligand termed urocontrin, i.e. [Bip⁴]URP, that is able to reduce the ex vivo efficacy of hUII- but not URP-induced vasoconstriction in rat aortic rings. In vivo studies support the pharmacological profile described above. Although urocontrin exert some residual agonist activity, this compound should be useful for the rational design of potent molecules that would allow discriminating specific biological action mediated by UII or URP.

Keywords: Abbreviations; SDS; sodium dodecylsulfate; RP-HPLC; reversed-phase high performance liquid chromatography; MALDI-TOF-MS; matrix-assisted desorption ionization time-of-flight mass spectrometry; Bip; (; S; )-2-amino-3-(1,1′-bipheny-4-yl) propanoic acidUrotensin II; Urotensin II-related peptide; Insurmountable antagonism; Urocontrin; Human UT receptors; Aortic ring bioassay

Inhibition of Rho kinases increases directional motility of microvascular endothelial cells by Johannes Breyer; Jana Samarin; Margot Rehm; Lena Lautscham; Ben Fabry; Margarete Goppelt-Struebe (pp. 616-626).

Rho kinases are major regulators of actin cytoskeletal organization and cell motility. Depending on the model system, inhibitors of Rho kinases (ROCK) have been reported to increase or decrease endothelial cell migration. In the present study we investigated the effect of Rho kinase inhibitors on microvascular endothelial cell migration with a special focus on the isoform ROCK2.Migration of microvascular endothelial cells was analyzed in a wound-healing, a spheroid-on-collagen migration assay and in cells embedded in collagen-1 gels. The non-selective Rho kinase inhibitor H1152 was compared to the selective ROCK2 inhibitor SLX2119 and to siRNA knock down.Non-selective inhibition of Rho kinases decreased cell-spanning F-actin fibers, loosened cell–cell contacts visualized by VE cadherin staining, and reduced cell–matrix interactions as shown by reduced Hic-5 expression in focal contacts. Rho kinase inhibitors facilitated directed migration of endothelial cells away from spheroids on fibronectin-coated plates and in collagen-1 gels. By contrast, migration of firmly attached endothelial cells, resembling intact vessels, was not promoted by Rho kinase inhibition. Selective inhibition of ROCK2 mimicked the cytoskeletal effects of H1152 and also increased cell motility, although to a lesser extent.In summary, Rho kinase inhibition enhanced the migration and cytoskeletal restructuring preferentially in freshly attached endothelial cells. ROCK2 may be a potential target to manipulate endothelial cell migration after vessel injury.

Keywords: Abbreviations; H1152; (; S; )-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-homopiperazine; BrdU; 5-bromo-2′-deoxyuridine; CTGF; connective tissue growth factor; DMOG; dimethyl-oxalylglycine; HUVEC; human umbilical vein endothelial cell; LPA; lysophosphatidic acid; MLC; myosin light chain; MYPT; MLC phosphatase; ROCK; Rho kinase, small G protein Rho-associated kinase; VEGF; vascular endothelial growth factor; Y-27632; (+)-(; R; )-; trans; -4-(1-aminoethyl)-; N; -(4-pyridyl) cyclohexanecarboxamide dihydrochlorideMicrovascular endothelial cells; Rho kinase; ROCK2; Connective tissue growth factor; Actin cytoskeleton; Cell motility

Identification of a new stilbene-derived inducer of paraoxonase 1 and ligand of the Aryl hydrocarbon Receptor by Erwan Guyot; Xavier Coumoul; Jean-François Chassé; Farid Khallouki; Jean François Savouret; Marc Poirot; Robert Barouki (pp. 627-632).

Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme, synthesized in the liver and secreted into the blood. PON1 displays antioxidant properties and is involved in organophosphorous compounds and oxidized lipids degradation. Because of these beneficial effects, pharmacological regulation of PON1 appears to be highly relevant in toxicology and cardiology. Recent studies undertaken on the regulation of the PON1 promoter in our laboratory have identified resveratrol, through its activation of the Aryl hydrocarbon Receptor (AhR), as a putative inducer of PON1. We have tested a new modulator of AhR, ( Z)-2,3-bis (4-nitrophenyl)-acrylonitrile, and established that it is a more potent inducer of PON1 at the mRNA, protein and enzymatic activity as compared to resveratrol. It also acts by activating the AhR. However, in contrast with traditional AhR agonists, it does not induce cyp1A1 transcription. ( Z)-2,3-bis (4-nitrophenyl)-acrylonitrile is therefore a specific AhR modulator targeting PON1.

Keywords: Paraoxonase 1; Resveratrol; Aryl hydrocarbon Receptor; Stilbene structure; Screening

Cellular bioenergetics is regulated by PARP1 under resting conditions and during oxidative stress by Katalin Módis; Domokos Gerő; Katalin Erdélyi; Petra Szoleczky; Douglas DeWitt; Csaba Szabo (pp. 633-643).

(a) During resting conditions, PARP1 regulates reserve respiratory capacity. (b) During oxidative stress, PARP1 becomes activated, leading to a reduction in cellular NAD+ and ATP pools, culminating in cell dysfunction.The goal of the current studies was to elucidate the role of the principal poly(ADP-ribose)polymerase isoform, PARP1 in the regulation of cellular energetics in endothelial cells under resting conditions and during oxidative stress.We utilized bEnd.3 endothelial cells and A549 human transformed epithelial cells. PARP1 was inhibited either by pharmacological inhibitors or by siRNA silencing. The Seahorse XF24 Extracellular Flux Analyzer was used to measure indices of mitochondrial respiration (oxygen consumption rate) and of glycolysis (extracellular acidification rate). Cell viability, cellular and mitochondrial NAD⁺ levels and mitochondrial biogenesis were also measured.Silencing of PARP1 increased basal cellular parameters of oxidative phosphorylation, providing direct evidence that PARP1 is a regulator of mitochondrial function in resting cells. Pharmacological inhibitors of PARP1 and siRNA silencing of PARP1 protected against the development of mitochondrial dysfunction and elevated the respiratory reserve capacity in endothelial and epithelial cells exposed to oxidative stress. The observed effects were unrelated to an effect on mitochondrial biogenesis. Isolated mitochondria of A549 human transformed epithelial cells exhibited an improved resting bioenergetic status after stable lentiviral silencing of PARP1; these effects were associated with elevated resting mitochondrial NAD⁺ levels in PARP1 silenced cells.PARP1 is a regulator of basal cellular energetics in resting endothelial and epithelial cells. Furthermore, endothelial cells respond with a decrease in their mitochondrial reserve capacity during low-level oxidative stress, an effect, which is attenuated by PARP1 inhibition. While PARP1 is a regulator of oxidative phosphorylation in resting and oxidatively stressed cells, it only exerts a minor effect on glycolysis.

Keywords: Abbreviations; 5-AIQ; 5-aminoisoquinolinone hydrochloride; DMEM; Dulbecco's Eagle modified medium; ECAR; extracellular acidification rate; ECL; enhanced chemiluminescence; FBS; fetal bovine serum; FCCP; carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone; H; ₂; O; ₂; hydrogen peroxide; INT; 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride; LDH; lactate dehydrogenase; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAD; ⁺; nicotinamide adenine dinucleotide; OCR; oxygen consumption rate; OXPHOS; oxidative phosphorylation; PARP1; poly(ADP-ribose) polymerase 1; PBS; phosphate buffered saline; PJ34; N-(-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide HCl; PMS; N-methylphenazonium methyl sulfate; PVDF; polyvinylidene fluoride; RNS; reactive nitrogen species; ROS; reactive oxygen species; SDS; sodium dodecyl sulfateOxidative stress; Poly(ADP)ribose polymerase; Mitochondrial bioenergetics; Oxidative phosphorylation; Intracellular NAD; ⁺; content; Respiratory reserve capacity

A truncated analogue of CCL2 mediates anti-fibrotic effects on murine fibroblasts independently of CCR2 by Christina Kalderén; Margareta Forsgren; Ulla Karlström; Karin Stefansson; Robert Svensson; Magnus M. Berglund; Gunnar Palm; Martin Selander; Maj Sundbom; Joakim Nilsson; Annelie Sjögren; Kristina Zachrisson; Stefan Svensson Gelius (pp. 644-652).

N-terminally truncated CCL2 [1–9+76], also named 7ND, was shown to inhibit secretion of extracellular matrix proteins, e.g. collagen I and fibronectin in fibroblasts isolated from lungs of bleomycin-induced mice.The truncated [1+9–76] CCL2 analogue, also known as 7ND, has been described in numerous reports as an anti-inflammatory and anti-fibrotic agent in a wide spectrum of animal models, e.g. models of cardiovascular disease, graft versus host disease and bleomycin-induced pulmonary fibrosis. 7ND has been reported to function as a competitive inhibitor of CCL2 signaling via CCR2 in human in vitro systems. In contrast, the mechanistic basis of 7ND action in animal models has not been previously reported. Here we have studied how 7ND interacts with CCL2 and CCR2 of murine origin. Surprisingly, 7ND was shown to be a weak inhibitor of murine CCL2/CCR2 signaling and displaced murine CCL2 (JE) from the receptor with a K_i>1μM. Using surface plasmon resonance, we found that 7ND binds murine CCL2 with a K_d of 670nM, which may indicate that 7ND inhibits murine CCL2/CCR2 signaling by a dominant negative mechanism rather than by competitive binding to the CCR2 receptor. In addition we observed that sub-nanomolar levels of 7ND mediate anti-fibrotic effects in CCR2 negative fibroblasts cultured from fibrotic lung of bleomycin-induced mice. Basal levels of extracellular matrix proteins were reduced (collagen type 1 and fibronectin) as well as expression levels of α-smooth muscle actin and CCL2. Our conclusion from these data is that the previously reported effects of 7ND in murine disease models most probably are mediated via mechanisms independent of CCR2.

Keywords: 7ND; CCL2; CCR2; Chemokine; Fibrosis

Modulation of AT-1R/AMPK-MAPK cascade plays crucial role for the pathogenesis of diabetic cardiomyopathy in transgenic type 2 diabetic (Spontaneous Diabetic Torii) rats by Arun Prasath Lakshmanan; Meilei Harima; Vijayakumar Sukumaran; Vivian Soetikno; Rajarajan Amirthalingam Thandavarayan; Kenji Suzuki; Makoto Kodama; Masaki Nagata; Ritsuo Takagi; Kenichi Watanabe (pp. 653-660).

There are evidences that the activation of AMPK is playing pivotal role in the lipid and glucose metabolism. It has been reported that both the AMPK and angiotensin-II acts as a negative regulator for each protein. It has been well proven that the MAPK cascade could be modulated by the presence of angiotensin-II. Moreover, studies were shown that p38 MAPK stimulates glucose uptake through the AMPK activation. Therefore, we speculate and tried to demonstrate that the modulation of AT-R/MAPK pathway through AMPK might play crucial roles for the pathogenesis of diabetic cardiomyopathy, using the transgenic (Spontaneous Diabetic Torii – SDT) rats. We performed Western blot analysis for the measurement of myocardial AT-R, AMPK and MAPK cascades-related protein expressions, p67-phox and caspase-12. In addition, we employed dihydroethidium (DHE), Azan Mallory and hemotoxylin eosin (HE) staining methods to demonstrate the superoxide radical production, fibrosis and hypertrophy, respectively. The protein expressions, such as AT-1R, p-ERK1/2, p67-phox and caspase-12 were found to be significantly increased and conversely, the Ang-(1-7) mas R, Tak1, LKB1 and p-AMPKα1, p-p38 MAPK and p-JNK protein expressions were found to be considerably decreased in the SDT rats, in comparison to the normal rats. The DHE, Azan Mallory and HE stainings also revealed that the SDT rats have more superoxide radical production, fibrosis and hypertrophy, respectively than the normal rats. Taken together, it is suggested that the modulation of AT-1R/AMPK-MAPK pathway might play crucial roles for the pathogenesis of diabetic cardiomyopathy and it could become an important therapeutic target to ameliorate the diabetic cardiomyopathy.

Keywords: Abbreviations; ACE-2; angiotensin converting enzyme-2; AMPK; AMP-activated protein kinase; Ang-(1-7); mas; R; angiotensin-(1-7); mas; receptor; Ang-II; angiotensin-II; AT-1R; Ang-II type 1 receptor; CVP; central venous pressure; DHE; dihydroethidium; ±d; p; /d; t; rate of intra-ventricular pressure rise and decline; EF; ejection fraction; ERK1/2; extracellular signal regulated kinase 1/2; FS; fractional shortening; HE; hemotoxylin eosin; IVSd; intra-ventricular septum thickness in diastole; IR; insulin resistance; JNK; c-Jun-N-terminal kinase; LKB1; serine/threonine kinase 11; LVDd; left ventricular dimension in diastole; LVDs; left ventricular dimension in systole; LVEDP; left ventricular end-diastole pressure; LVP; left ventricular pressure; LVPWd; left ventricular posterior wall thickness in diastole; MAPK; mitogen activated protein kinase; mmHg; millimeter mercury; mmHg/s; millimeter mercury/second; p38 MAPK; p38 mitogen activated protein kinase; p-AMPKα1; phospho-AMP-activated protein kinase alpha1; p-ERK1/2; phospho- extracellular signal regulated kinase 1/2; p-JNK; phospho-c-Jun-N-terminal kinase; p-p38 MAPK; phospho-p38 mitogen activated protein kinase; RAS; rennin-angiotensin system; SD; Sprague–Dawley; SDT; Spontaneous Diabetic Torii; STZ; streptozotocin; Tak1; transforming growth factor-beta activated kinase 1Spontaneous Diabetic Torii; Diabetic cardiomyopathy; AMPK; AT-1R; MAPK

Interleukin-17A increases leptin production in human bone marrow mesenchymal stem cells by Minsoo Noh (pp. 661-670).

Leptin production in extra-adipose tissues like hBM-MSCs and osteoblasts in response to IL-17A and dexamethasone.Lineage commitment of human bone marrow mesenchymal stem cells (hBM-MSCs) to adipocytes or osteoblasts has been suggested as a model system to study the relationship between type II diabetes and abnormal bone metabolism. Leptin and IL-17A inhibit adipogenesis whereas they promote osteogenesis in MSCs. Due to pathophysiologic roles of IL-17A in human metabolic diseases and bone metabolism, it was evaluated whether IL-17A-dependent inverse regulation on adipogenesis and osteogenesis was related to endogenous leptin production in hBM-MSCs. In the analysis of adiponectin and leptin secretion profiles of hBM-MSCs in response to various combinations of differentiation inducing factors, it was found that dexamethasone, a common molecule used for both adipogenesis and osteogenesis, increased leptin production in hBM-MSCs. Importantly, the level of leptin production during osteogenesis in hBM-MSCs was higher than that during adipogenesis, implicating a significant leptin production in extra-adipose tissues. IL-17A increased leptin production in hBM-MSCs and also under the condition of osteogenesis. In spite of direct inhibition on adipogenesis, IL-17A up-regulated leptin production in hBM-MSC-derived adipocytes. Anti-leptin antibody treatment partially antagonized the IL-17A dependent inhibition of adipogenesis in hBM-MSCs, suggesting a role of leptin in mediating the inverse regulation of IL-17A on osteogenesis and adipogenesis in hBM-MSCs. Therefore, the IL-17A-induced leptin production may provide a key clue to understand a molecular mechanism on the lineage commitment of hBM-MSCs into adipocytes or osteoblasts. In addition, leptin production in extra-adipose tissues like MSCs and osteoblasts should be considered in future studies on leptin-associated human diseases.

Keywords: Leptin; IL-17A; Human bone marrow mesenchymal stem cells; Adipogenesis; Osteogenesis

Molecular effects of gallium on osteoclastic differentiation of mouse and human monocytes by E. Verron; A. Loubat; G.F. Carle; C. Vignes-Colombeix; I. Strazic; J. Guicheux; N. Rochet; J.M. Bouler; J.C. Scimeca (pp. 671-679).

We had previously reported that gallium (Ga) inhibited both the differentiation and resorbing activity of osteoclasts in a dose-dependent manner. To provide new insights into Ga impact on osteoclastogenesis, we investigated here the molecular mechanisms of Ga action on osteoclastic differentiation of monocytes upon Rankl treatment. We first observed that Ga treatment inhibited the expression of Rankl-induced early differentiation marker genes, while the same treatment performed subsequently did not modify the expression of late differentiation marker genes. Focusing on the early stages of osteoclast differentiation, we observed that Ga considerably disturbed both the initial induction as well as the autoamplification step of Nfatc1 gene. We next demonstrated that Ga strongly up-regulated the expression of Traf6, p62 and Cyld genes, and we observed concomitantly an inhibition of IκB degradation and a blockade of NFκB nuclear translocation, which regulates the initial induction of Nfatc1 gene expression. In addition, Ga inhibited c-Fos gene expression, and subsequently the auto-amplification stage of Nfatc1 gene expression. Lastly, considering calcium signaling, we observed upon Ga treatment an inhibition of calcium-induced Creb phosphorylation, as well as a blockade of gadolinium-induced calcium entry through TRPV-5 calcium channels. We identify for the first time Traf6, p62, Cyld, IκB, NFκB, c-Fos, and the calcium-induced Creb phosphorylation as molecular targets of Ga, this tremendously impacting the expression of the master transcription factor Nfatc1. In addition, our results strongly suggest that the TRPV-5 calcium channel, which is located within the plasma membrane, is a target of Ga action on human osteoclast progenitor cells.

Keywords: Gallium; Osteoclast; CD11b; ⁺; NFATc1; NFκB; Calcium

Dopamine-₃ receptor modulates intraocular pressure: Implications for glaucoma by Claudio Bucolo; Gian Marco Leggio; Adriana Maltese; Alessandro Castorina; Velia D’Agata; Filippo Drago (pp. 680-686).

The aim of the present study was to investigate the role of D₃ receptor on intraocular pressure regulation using WT and KO D₃R^−/− mice. Both mice were used with normal eye pressure or steroid-induced ocular hypertension. As measured by tonometry, the topical application of 7-OH-DPAT, a dopamine D₃-preferring receptor agonist, significantly decreased, in a dose-dependent manner, the intraocular pressure in WT mice both in an ocular normotensive group and an ocular hypertensive group. Pretreatment with U-99194A, a D₃ receptor antagonist, reverted 7-OH-DPAT induced ocular hypotension in WT mice. No change of intraocular pressure was observed after topical application of 7-OH-DPAT in KO D₃R^−/− mice. PCR analysis demonstrated the presence of all dopamine receptor genes in eye tissues obtained from WT mice, and the lack of D₃R mRNAs in KO mice. The present study identified the D₃R subtype as the most important receptor of the dopaminergic system to modulate intraocular pressure with relevant implications for glaucoma that represents one of the most crippling optic neuropathies.

Keywords: Dopamine-; ₃; receptor; Intraocular pressure; Optic neuropathy; Glaucoma

The effect of sodium selenite on liver growth and thioredoxin reductase expression in regenerative and neoplastic liver cell proliferation by Suvd Erkhembayar; Annelie Mollbrink; Lennart C. Eriksson (pp. 687-693).

Sodium selenite inhibits growth of liver tumours but not liver regeneration after partial hepatectomy (PH). An experimental rat study.Selenium in supra-nutritional doses is tumour-preventative in animal models and in humans. In this work, we have compared the effect of sodium selenite on tumour growth in a rat hepatocarcinogenesis model with the effect of sodium selenite on the regeneration of liver mass after partial hepatectomy. In the tumour model, 5μg/mL sodium selenite in the drinking water reduced the rate of tumour growth for up to 12 months after initiation; the volume fraction of liver cancers was 14±4% with a mean bromodeoxyuridine-index of 19±11% in the treated rats compared to a volume fraction of 26±7% with a mean bromodeoxyuridine-index of 42±27% in the control rats. Despite its efficacy in reducing tumour growth, 5μg/mL sodium selenite treatment did not affect the gain of liver mass or the rate of cell proliferation after partial hepatectomy. In the regenerating livers, the activity of the cytosolic selenoenzyme thioredoxin reductase (TrxR1) was briefly and transiently increased, an increase further potentiated by sodium selenite. TrxR1 was selectively over expressed in proliferating liver tumours in relation to the surrounding liver tissue in the tumour model, as shown using immunohistochemistry analyses. We suggest that sodium selenite is a suitable candidate for liver cancer prevention in patients with chronic liver diseases that are dependent on sustained liver regeneration due to its differential effects on neoplastic and regenerative cell proliferation. Furthermore, the over expression of TrxR1 in liver neoplasia can only partly be explained by increased growth.

Keywords: Sodium selenite; Rat liver cancer; Liver regeneration; Tumour prevention; Thioredoxin reductase 1

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Biochemical Pharmacology (v.83, #5)