Tian-Quan Cai

11β-Hydroxysteroid Dehydrogenase Type 1 Is Induced in Human Monocytes upon Differentiation to Macrophages

11β-hydroxysteroid dehydrogenases (11β-HSD) perform prereceptor metabolism of glucocorticoids through interconversion of the active glucocorticoid, cortisol, with inactive cortisone. Although the immunosuppressive and anti-inflammatory activities of glucocorticoids are well documented, the expression of 11β-HSD enzymes in immune cells is not well understood. Here we demonstrate that 11β-HSD1, which converts cortisone to cortisol, is expressed only upon differentiation of human monocytes to macrophages. 11β-HSD1 expression is concomitant with the emergence of peroxisome proliferator activating receptor γ, which was used as a surrogate marker of monocyte differentiation. The type 2 enzyme, 11β-HSD2, which converts cortisol to cortisone, was not detectable in either monocytes or cultured macrophages. Incubation of monocytes with IL-4 or IL-13 induced 11β-HSD1 activity by up to 10-fold. IFN-γ, a known functional antagonist of IL-4 and IL-13, suppressed the induction of 11β-HSD1 by these cytokines. THP-1 cells, a human macrophage-like cell line, expressed 11β-HSD1 and low levels of 11β-HSD2. The expression of 11β-HSD1 in these cells is up-regulated 4-fold by LPS. In summary, we have shown strong expression of 11β-HSD1 in cultured human macrophages and THP-1 cells. The presence of the enzyme in these cells suggests that it may play a role in regulating the immune function of these cells.

Statins suppress THP-1 cell migration and secretion of matrix metalloproteinase 9 by inhibiting geranylgeranylation

Macrophages secrete matrix metalloproteinase 9 (MMP‐9), an enzyme that weakens the fibrous cap of atherosclerotic plaques, predisposing them to plaque rupture and subsequent ischemic events. Recent work indicates that statins strongly reduce the possibility of heart attack. Furthermore, these compounds appear to exert beneficial effects not only by lowering plasma low‐density‐lipoprotein cholesterol but also by directly affecting the artery wall. To evaluate whether statins influence the proinflammatory responses of monocytic cells, we studied their effects on the chemotactic migration and MMP‐9 secretion of human monocytic cell line THP‐1. Simvastatin dose dependently inhibited THP‐1 cell migration mediated by monocyte chemoattractant protein 1, with a 50% inhibitory concentration of about 50 nM. It also inhibited bacterial lipopolysaccharide‐stimulated secretion of MMP‐9. The effects of simvastatin were completely reversed by mevalonate and its derivatives, farnesylpyrophosphate and geranylgeranyl pyrophosphate, but not by ubiquinone. Additional studies revealed similar but more profound inhibitory effects with L‐839,867, a specific inhibitor of geranylgeranyl transferase. However, α‐hydroxyfarnesyl phosphonic acid, an inhibitor of farnesyl transferase, had no effect. C3 exoenzyme, a specific inhibitor of the prenylated small signaling Rho proteins, mimicked the inhibitory effects of simvastatin and L‐839,867. These data supported the role of geranylgeranylation in the migration and MMP‐9 secretion of monocytes.

Role of GPR81 in lactate-mediated reduction of adipose lipolysis.

Heavy exercise or oxygen deficit often links with higher levels of arterial lactate and lower levels of plasma free fatty acids (FFA). Treatment with lactate reduces circulating levels of FFA in vivo and lipolysis in adipose tissues in vitro. However, the underlying mechanism has remained unclear. Here we employ pharmacological and genetic approaches to show that GPR81, an orphan G-protein-coupled receptor with relatively restricted expression in the adipose tissues, functions as a receptor for lactate and can mediate an anti-lipolytic effect of lactate. GPR81 may thus function as a sensor of lactate that can modulate the FFA pool under exercise or conditions of oxygen deficit.

Induction of 11β-hydroxysteroid dehydrogenase type 1 but not -2 in human aortic smooth muscle cells by inflammatory stimuli

The 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes catalyze the interconversion of active glucocorticoids (GC) with their inert metabolites, thereby regulating the functional activity of GC. While 11beta-HSD type 1 (11beta-HSD1) activates GC from their 11-keto metabolites, 11beta-HSD type 2 (11beta-HSD2) inactivates GC. Here we report that both of these enzymes are expressed in human aortic smooth muscle cells (SMC), and that 11beta-HSD1 is more abundant and is differentially regulated relative to 11beta-HSD2. Stimulation of SMC with IL-1beta or TNFalpha led to a time- and dose-dependent increase of mRNA levels for 11beta-HSD1, while 11beta-HSD2 mRNA levels decreased. Parallel enzyme activity studies showed increased conversion of 3H-cortisone to 3H-cortisol but not 3H-cortisol to 3H-cortisone, demonstrating 11beta-HSD1 in SMC acts primarily as a reductase. A similar increase of 11beta-HSD1 mRNA expression was also found in human bronchial SMC upon stimulation, indicating the regulatory effect is not limited to vascular smooth muscle. Additional parallel studies revealed a similar pattern of induction for 11beta-HSD1 and monocyte chemoattractant protein-1, a well-defined proinflammatory molecule. These data suggest 11beta-HSD1 may play an important role in regulating inflammatory responses in the artery wall and lung.

3-(1H-Tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclopentapyrazole (MK-0354): A Partial Agonist of the Nicotinic Acid Receptor, G-Protein Coupled Receptor 109a, with Antilipolytic but No Vasodilatory Activity in Mice

The discovery and profiling of 3-(1H-tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclopentapyrazole (5a, MK-0354), a partial agonist of GPR109a, is described. Compound 5a retained the plasma free fatty acid lowering effects in mice associated with GPR109a agonism, but did not induce vasodilation at the maximum feasible dose. Moreover, preadministration of 5a blocked the flushing effect induced by nicotinic acid but not that induced by PGD2. This profile made 5a a suitable candidate for further study for the treatment of dyslipidemia.

MMP-2 mediates ethanol-induced invasion of mammary epithelial cells over-expressing ErbB2

Ethanol is a tumor promoter and may enhance the metastasis of breast cancer. We have previously demonstrated that over‐expression of ErbB2 promoted ethanol‐mediated invasion of mammary epithelial cells and breast cancer cells. However, the underlying cellular/molecular mechanisms remain unknown. By gelatin zymography, we showed that over‐expression of ErbB2 increased the production of matrix metalloproteinase‐2 (MMP‐2) and MMP‐9 in human mammary epithelial cells (HB2). Transient or stable transfection of ErbB2 cDNA to HB2 cells upregulated the transcripts and the activity of the MMP‐2/‐9 gene promoter; the upregulation of MMP‐2/‐9 expression was mediated by p38 mitogen‐activated protein kinase (p38 MAPK) and phosphatidylinositol 3‐kinase (PI3K). Although ethanol, at physiologically relevant concentrations (100–400 mg/dl), did not affect the production of MMP‐2/‐9, it activated MMP‐2 in HB2 cells over‐expressing ErbB2 (HB2ErbB2), but not HB2 cells; it enhanced the cleavage of proform MMP‐2 (72 kDa) to an active form (62 kDa). The activation was dependent on c‐jun N‐terminal kinases (JNKs) and reactive oxygen species (ROS). On the other hand, ethanol affected neither the expression nor the activation of MMP‐9. Selective inhibitors of MMP‐2 (SB‐3CT and OA‐Hy) and antioxidants significantly inhibited ethanol‐stimulated invasion of HB2ErbB2 cells. Furthermore, knocking down MMP‐2 by small interference RNA also induced a partial blockage on ethanol‐promoted invasion of HB2ErbB2 cells. Thus, ethanol‐stimulated invasion of cells over‐expressing ErbB2 was mediated, at least partially, by MMP‐2 activation.

Developmental expression of matrix metalloproteinases 2 and 9 and their potential role in the histogenesis of the cerebellar cortex

The development of the cerebellar cortex depends on intrinsic genetic programs and orchestrated cell–cell/cell–matrix interactions. Matrix metalloproteinases (MMPs) are proteolytic enzymes that play an important role in these interactions. MMP‐2 and MMP‐9 are involved in diverse neuronal functions including migration, process extension, and synaptic plasticity. We investigated the spatiotemporal pattern of expression/activity of MMP‐2/MMP‐9 in the developing cerebellum and their role in the histogenesis of the cerebellar cortex. The levels of transcripts of MMP‐2/MMP‐9 were measured with real‐time quantitative polymerase chain reaction. An initial decrease in MMP‐2/MMP‐9 transcripts was observed between postnatal days 3 (PD3) and PD6, and the mRNA levels remained relatively constant thereafter. Zymographic analysis revealed that the expression/activity of MMP‐2/MMP‐9 persisted longer than their transcripts; the downregulation occurred around PD9, suggesting a mechanism of translational or post‐translational regulation. The gelatinase activity was localized in the external granule layer (EGL) and the internal granule layer during PD3–PD12. The immunoreactivity of MMP‐2 was mainly localized in the EGL, the Bergmann glial fibers, and the Purkinje cell layer (PCL), whereas MMP‐9 immunoreactivity was detected intensively in the PCL and the extracellular space of the molecular layer. Expression of MMP‐9 was relatively weak in the EGL. The immunoreactivity of MMP‐2/MMP‐9 became undetectable after PD21. A similar expression pattern of MMP‐2/MMP‐9 was observed in organotypic cerebellar slice cultures. Exposure of organotypic slices to a specific MMP‐2/MMP‐9 inhibitor significantly increased the thickness of the EGL and concurrently decreased the number of migrating granule neurons in the molecular layer. Thus, MMP‐2 and MMP‐9 play a role in the postnatal cerebellar morphogenesis. J. Comp. Neurol. 481:403–415, 2005.

Production of matrix metalloproteinase-9 in CaCO-2 cells in response to inflammatory stimuli.

Matrix metalloproteinase-9 (MMP-9) may play an important role in the development of inflammatory bowel disease (IBD). However, the cellular source of MMP-9 in the inflamed mucosa of IBD remains unclear. Here we report that MMP-9 mRNA is expressed in CaCO-2 cells, an intestinal epithelial cell line, and that its expression is upregulated by inflammatory stimuli. Stimulation of CaCO-2 cells with interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha (TNF-alpha) led to a dose-dependent increase in expression and secretion of MMP-9. In contrast, bacterial lipopolysaccharide (LPS) failed to induce expression or secretion of MMP-9, suggesting that an inflammatory reaction leading to cytokine release is a necessary step for the induction of MMP-9 release in intestinal epithelial cells. Additional studies show that induction of MMP-9 mRNA peaked at 16 h of IL-1beta stimulation, whereas expression of monocyte chemoattractant protein-1 (MCP-1) and IL-8 both peaked at 3 h of stimulation. Treatment of CaCO-2 cells with rosiglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist, significantly reduced secretion of MMP-9, indicating that agents that activate PPAR-gamma may have therapeutic use in patients with IBD.

Discovery of novel tricyclic full agonists for the G-protein-coupled niacin receptor 109A with minimized flushing in rats.

Tricyclic analogues were rationally designed as the high affinity niacin receptor G-protein-coupled receptor 109A (GPR109A) agonists by overlapping three lead structures. Various tricyclic anthranilide and cycloalkene carboxylic acid full agonists were discovered with excellent in vitro activity. Compound 2g displayed a good therapeutic index regarding free fatty acids (FFA) reduction and vasodilation effects in rats, with very weak cytochrome P450 2C8 (CYP2C8) and cytochrome P450 2C9 (CYP2C9) inhibition, and a good mouse pharmacokinetics (PK) profile.

Discovery of a potent and selective small molecule hGPR91 antagonist

GPR91, a 7TM G-Protein-Coupled Receptor, has been recently deorphanized with succinic acid as its endogenous ligand. Current literature indicates that GPR91 plays role in various pathophysiology including renal hypertension, autoimmune disease and retinal angiogenesis. Starting from a small molecule high-throughput screening hit 1 (hGPR91 IC(50): 0.8 μM)-originally synthesized in Merck for Bradykinin B(1) Receptor (BK(1)R) program, systematic structure-activity relationship study led us to discover potent and selective hGPR91 antagonists e.g. 2c, 4c, and 5 g (IC(50): 7-35 nM; >1000 fold selective against hGPR99, a closest related GPCR; >100 fold selective in Drug Matrix screening). This initial work also led to identification of two structurally distinct and orally bio-available lead compounds: 5g (%F: 26) and 7e (IC(50): 180 nM; >100 fold selective against hGPR99; %F: 87). A rat pharmacodynamic assay was developed to characterize the antagonists in vivo using succinate induced increase in blood pressure. Using two representative antagonists, 2c and 4c, the GPR91 target engagement was subsequently demonstrated using the designed pharmacodynamic assay.