TGF-␤1 expression closely associates with activation and conversion of fibroblasts to a myofibroblast phenotype and synthesis of an alternatively spliced cellular fibronectin variant, Fn-ED-A. Reactive oxygen species (ROS), such as superoxide, which is a product of NAD(P)H oxidase, also promote the transition of fibroblasts to myofibroblasts, but whether these two pathways are interrelated is unknown. Here, we examined a role for NAD(P)H oxidase-derived ROS in TGF-␤1-induced activation of rat kidney fibroblasts and expression of ␣-smooth muscle actin (␣-SMA) and Fn-ED-A. In vitro, TGF-␤1 stimulated formation of abundant stress fibers and increased expression of both ␣-SMA and Fn-ED-A. In addition, TGF-␤1 increased both the activity of NADPH oxidase and expression of Nox2 and Nox4, homologs of the NAD(P)H oxidase family, indicating that this growth factor induces production of ROS. Small interfering RNA targeted against Nox4 markedly inhibited TGF-␤1-induced stimulation of NADPH oxidase activity and reduced ␣-SMA and Fn-ED-A expression. Inhibition of TGF-␤1 receptor 1 blocked Smad3 phosphorylation; reduced TGF-␤1-enhanced NADPH oxidase activity; and decreased expression of Nox4, ␣-SMA, and Fn-ED-A. Diphenyleneiodonium, an inhibitor of flavin-containing enzymes such as the Nox oxidases, had no effect on TGF-␤1-induced Smad3 but reduced both ␣-SMA and Fn-ED-A protein expression. The Smad3 inhibitor SIS3 reduced NADPH oxidase activity, Nox4 expression, and blocked ␣-SMA and Fn-ED-A, indicating that stimulation of myofibroblast activation by ROS is downstream of Smad3. In addition, TGF-␤1 stimulated phosphorylation of extracellular signal-regulated kinase (ERK1/2), and this was inhibited by blocking TGF-␤1 receptor 1, Smad3, or the Nox oxidases; ERK1/2 activation increased ␣-SMA and Fn-ED-A. Taken together, these results suggest that TGF-␤1-induced conversion of fibroblasts to a myofibroblast phenotype involves a signaling cascade through Smad3, NAD(P)H oxidase, and ERK1/2.

Binding of cocaine to the dopamine transporter (DAT) protein blocks synaptic dopamine clearance, triggering the psychoactive effects associated with the drug; the discrete drug-protein interactions, however, remain poorly understood. A longstanding postulate holds that cocaine inhibits DAT-mediated dopamine transport via competition with dopamine for formation of an ionic bond with the DAT transmembrane aspartic acid residue D79. In the present study, DAT mutations of this residue were generated and assayed for translocation of radiolabeled dopamine and binding of radiolabeled DAT inhibitors under identical conditions. When feasible, dopamine uptake inhibition potency and apparent binding affinity K i values were determined for structurally diverse DAT inhibitors. The glutamic acid substitution mutant (D79E) displayed values indistinguishable from wild-type DAT in both assays for the charge-neutral cocaine analog 8-oxa-norcocaine, a finding not supportive of the D79 "salt bridge" ligand-docking model. In addressing whether the D79 side chain contributes to the DAT binding sites of other portions of the cocaine pharmacophore, only inhibitors with modifications of the tropane ring C-3 substituent, i.e., benztropine and its analogs, displayed a substantially altered dopamine uptake inhibition potency as a function of the D79E mutation. A single conservative amino acid substitution thus differentiated structural requirements for benztropine function relative to those for all other classical DAT inhibitors. Distinguishing the precise mechanism of action of this DAT inhibitor with relatively low abuse liability from that of cocaine may be attainable using DAT mutagenesis and other structure-function studies, opening the door to rational design of therapeutic agents for cocaine abuse.Millions of individuals worldwide are addicted to cocaine, a public health crisis that also carries a substantial burden to society in the form of medical expenses, lost earnings, and increased crime associated with psychostimulant abuse. Although therapeutics including buprenorphine and methadone are clinically accessible to treat opiate abuse and addiction, no such Food and Drug Administration-approved medication is available for the treatment of cocaine addiction. Pharmacological and behavioral studies have established that the euphoric and addictive effects of cocaine are initiated by its binding to the dopamine transporter (DAT) protein, blocking clearance of dopamine from central nervous system synapses and thereby prolonging dopaminergic neurotransmission in brain areas associated with reward (Ritz et al., 1987). The DAT is therefore a logical target for development of medications for cocaine abuse. Elucidating the relationship between the DAT and its substrates and inhibitors is essential, especially regarding cocaine points of contact with the DAT protein relevant to its inhibition of dopamine uptake.Because dopamine and all classical DAT inhibitors possess a protonated nitrogen atom, a longstanding model for cocaine

In this study, the effects of the light/dark cycle, hormone replacement therapy (HRT), and nocturnal melatonin supplementation on osteogenic markers and serum melatonin levels were examined in a blind mouse model (MMTV-Neu transgenic mice). Melatonin levels in this mouse strain (FVB/N) with retinal degeneration (rd-/-) fluctuate in a diurnal manner, suggesting that these mice, although blind, still perceive light. Real-time RT-PCR analyses demonstrated that Runx2, Bmp2, Bmp6, Bglap, and Per2 mRNA levels coincide with melatonin levels. The effect of chronic HRT (0.5 mg 17β-estradiol + 50 mg progesterone in 1800 kcal of diet) alone and in combination with melatonin (15 mg/L drinking water) on bone quality and density was also assessed by histomorphometry and microcomputed tomography, respectively. Bone density was significantly increased (P < 0.05) after 1 yr of treatment with the individual therapies, HRT (22% increase) and nocturnal melatonin (20% increase) compared to control. Hormone replacement therapy alone also increased surface bone, decreased trabecular space, and decreased the number of osteoclasts without affecting osteoblast numbers compared to the control group (P < 0.05). Chronic HRT + melatonin therapy did not significantly increase bone density, even though this combination significantly increased Bglap mRNA levels. These data suggest that the endogenous melatonin rhythm modulates markers important to bone physiology. Hormone replacement therapy with or without nocturnal melatonin in cycling mice produces unique effects on bone markers and bone density. The effects of these therapies alone and combined may improve bone health in women in perimenopause and with low nocturnal melatonin levels from too little sleep, too much light, or age.

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Melatonin induces cellular differentiation in numerous cell types. Data show that multiple mechanisms are involved in these processes that are cell-type specific and may be receptor dependent or independent. The focus of this study was to specifically assess the role of human MT1 melatonin receptors in cellular differentiation using an MT1-Chinese hamster ovary (CHO) model; one that reproducibly produces measurable morphologic changes in response to melatonin. Using multiple approaches, we show that melatonin induces MT1-CHO cells to hyperelongate through a MEK 1/2, and ERK 1/2-dependent mechanism that is dependent upon MT1 receptor internalization, Gi protein activation, and clathrin-mediated endocytosis. Using immunoprecipitation analysis, we show that MT1 receptors form complexes with Gi(alpha) 2,3, Gq(alpha), beta-arrestin-2, MEK 1/2, and ERK 1/2 in the presence of melatonin. We also show that MEK and ERK activity that is induced by melatonin is dependent on Gi protein activation, clathrin-mediated endocytosis and is modulated by microtubules. We conclude from these studies that melatonin-induced internalization of human MT1 melatonin receptors in CHO cells is responsible for activating both MEK 1/2 and ERK 1/2 to drive these morphologic changes. These events, as mediated by melatonin, require Gi protein activation and endocytosis mediated through clathrin, to form MT1 receptor complexes with beta-arrestin-2/MEK 1/2 and ERK 1/2. The MT1-CHO model is invaluable to mapping out signaling cascades as mediated through MT1 receptors especially because it separates out MEK/ERK 1/2 activation by MT1 receptors from that of receptor tyrosine kinases.