Pharmacological targeting of glutamatergic neurons within the brainstem for weight reduction.

Food intake and body weight are tightly regulated by neurons within specific brain regions, including the brainstem, where acute activation of dorsal raphe nucleus (DRN) glutamatergic neurons expressing the glutamate transporter Vglut3 (DRNVglut3) drive a robust suppression of food intake and enhance locomotion. Activating Vglut3 neurons in DRN suppresses food intake and increases locomotion, suggesting that modulating the activity of these neurons might alter body weight. Here, we show that DRNVglut3 neurons project to the lateral hypothalamus (LHA), a canonical feeding center that also reduces food intake. Moreover, chronic DRNVglut3 activation reduces weight in both leptin-deficient (ob/ob) and leptin-resistant diet-induced obese (DIO) male mice. Molecular profiling revealed that the orexin 1 receptor (Hcrtr1) is highly enriched in DRN Vglut3 neurons, with limited expression elsewhere in the brain. Finally, an orally bioavailable, highly selective Hcrtr1 antagonist (CVN45502) significantly reduces feeding and body weight in DIO. Hcrtr1 is also co-expressed with Vglut3 in the human DRN, suggesting that there might be a similar effect in human. These results identify a potential therapy for obesity by targeting DRNVglut3 neurons while also establishing a general strategy for developing drugs for central nervous system disorders.

Melanocortin 4 receptor stimulation prevents antidepressant-associated weight gain in mice caused by long-term fluoxetine exposure.

Contrasting with the predicted anorexigenic effect of increasing brain serotonin signaling, long-term use of selective serotonin reuptake inhibitor (SSRI) antidepressants correlates with body weight (BW) gain. This adverse outcome increases the risk of transitioning to obesity and interferes with treatment compliance. Here, we show that orally administered fluoxetine (Flx), a widely prescribed SSRI, increased BW by enhancing food intake in healthy mice at 2 different time points and through 2 distinct mechanisms. Within hours, Flx decreased the activity of a subset of brainstem serotonergic neurons by triggering autoinhibitory signaling through 5-hydroxytryptamine receptor 1a (Htr1a). Following a longer treatment period, Flx blunted 5-hydroxytryptamine receptor 2c (Htr2c) expression and signaling, decreased the phosphorylation of cAMP response element-binding protein (CREB) and STAT3, and dampened the production of pro-opiomelanocortin (POMC, the precursor of α-melanocyte stimulating hormone [α-MSH]) in hypothalamic neurons, thereby increasing food intake. Accordingly, exogenous stimulation of the melanocortin 4 receptor (Mc4r) by cotreating mice with Flx and lipocalin 2, an anorexigenic hormone signaling through this receptor, normalized feeding and BW. Flx and other SSRIs also inhibited CREB and STAT3 phosphorylation in a human neuronal cell line, suggesting that these noncanonical effects could also occur in individuals treated long term with SSRIs. By defining the molecular basis of long-term SSRI-associated weight gain, we propose a therapeutic strategy to counter this effect.

Serotonin-reuptake inhibitors act centrally to cause bone loss in mice by counteracting a local anti-resorptive effect.

The use of selective serotonin-reuptake inhibitors (SSRIs) has been associated with an increased risk of bone fracture, raising concerns about their increasingly broader usage. This deleterious effect is poorly understood, and thus strategies to avoid this side effect remain elusive. We show here that fluoxetine (Flx), one of the most-prescribed SSRIs, acts on bone remodeling through two distinct mechanisms. Peripherally, Flx has anti-resorptive properties, directly impairing osteoclast differentiation and function through a serotonin-reuptake-independent mechanism that is dependent on intracellular Ca2+ levels and the transcription factor Nfatc1. With time, however, Flx also triggers a brain-serotonin-dependent rise in sympathetic output that increases bone resorption sufficiently to counteract its local anti-resorptive effect, thus leading to a net effect of impaired bone formation and bone loss. Accordingly, neutralizing this second mode of action through co-treatment with the ß-blocker propranolol, while leaving the peripheral effect intact, prevents Flx-induced bone loss in mice. Hence, this study identifies a dual mode of action of SSRIs on bone remodeling and suggests a therapeutic strategy to block the deleterious effect on bone homeostasis from their chronic use.

Simplified microenvironments and reduced cell culture size influence the cell differentiation outcome in cellular microarrays.

Cellular microarrays present a promising tool for multiplex evaluation of the signalling effect of substrate-immobilized factors on cellular differentiation. In this paper, we compare the early myoblast-to-osteoblast cell commitment steps in response to a growth factor stimulus using standard well plate differentiation assays or cellular microarrays. Our results show that restraints on the cell culture size, inherent to cellular microarrays, impair the differentiation outcome. Also, while cells growing on spots with immobilised BMP-2 are early biased towards the osteoblast fate, longer periods of cell culturing in the microarrays result in cell proliferation and blockage of osteoblast differentiation. The results presented here raise concerns about the efficiency of cell differentiation when the cell culture dimensions are reduced to a simplified microspot environment. Also, these results suggest that further efforts should be devoted to increasing the complexity of the microspots composition, aiming to replace signalling cues missing in this system.

Osterix induces Col1a1 gene expression through binding to Sp1 sites in the bone enhancer and proximal promoter regions.

Bone-specific transcription factors promote differentiation of mesenchymal precursors toward the osteoblastic cell phenotype. Among them, Runx2 and Osterix have been widely accepted as master osteogenic factors, since neither Runx2 nor Osterix null mice form mature osteoblasts. Recruitment of Osterix to a number of promoters of bone-specific genes has been shown. However, little is known about the functional interactions between Osterix and the Col1a1 promoter. In this study we determined in several mesenchymal and osteoblastic cell types that either BMP-2 or Osterix overexpression increased Col1a1 transcription. We identified consensus Sp1 sequences, located in the proximal promoter and in the bone-enhancer, as Osterix binding regions in the Col1a1 promoter in vitro and in vivo. Furthermore, we show that p38 or Erk MAPK signaling is required for maximal transcriptional effects on Col1a1 expression. Runx2 has been shown to activate Col1a1 expression through binding to sites which are located close to the Sp1 sites where Osterix binds. Our data show that overexpression of Runx2 and Osterix leads to a cooperative effect on the expression of the Col1a1 endogenous gene and its promoter reporter construct. These effects mainly affect the long isoform of Osterix which suggest that the two Osterix isoforms might display some differential effects on the transactivation of bone-specific genes.

Noncanonical BMP signaling regulates cyclooxygenase-2 transcription.

Activation of p38 MAPK has been shown to be relevant for a number of bone morphogenetic protein (BMP) physiological effects. We report here the involvement of noncanonical phosphorylated mothers against decapentaplegic (Smad) signaling in the transcriptional induction of Cox2 (Ptgs2) by BMP-2 in mesenchymal cells and organotypic calvarial cultures. We demonstrate that different regulatory elements are required for regulation of Cox2 expression by BMP-2: Runt-related transcription factor-2 and cAMP response element sites are essential, whereas a GC-rich Smad binding element is important for full responsiveness. Efficient transcriptional activation requires cooperation between transcription factors because mutation of any element results in a strong decrease of BMP-2 responsiveness. BMP-2 activation of p38 leads to increased recruitment of activating transcription factor-2, Runx2, Smad, and coactivators such as p300 at the responsive sites in the Cox2 proximal promoter. We demonstrate, by either pharmacological or genetic analysis, that maximal BMP-2 effects on Cox2 and JunB expression require the function of p38 and its downstream effector mitogen/stress-activated kinase 1. Altogether our results strongly suggest that cooperative effects between canonical and noncanonical BMP signaling allow the fine-tuning of BMP transcriptional responses on specific target genes.

p38 regulates expression of osteoblast-specific genes by phosphorylation of osterix.

Osterix, a zinc finger transcription factor, is specifically expressed in osteoblasts and osteocytes of all developing bones. Because no bone formation occurs in Osx-null mice, Osterix is thought to be an essential regulator of osteoblast differentiation. We report that, in several mesenchymal and osteoblastic cell types, BMP-2 induces an increase in expression of the two isoforms of Osterix arising from two alternative promoters. We identified a consensus Sp1 sequence (GGGCGG) as Osterix binding regions in the fibromodulin and the bone sialoprotein promoters in vitro and in vivo. Furthermore, we show that Osterix is a novel substrate for p38 MAPK in vitro and in vivo and that Ser-73 and Ser-77 are the regulatory sites phosphorylated by p38. Our data also demonstrate that Osterix is able to increase recruitment of p300 and Brg1 to the promoters of its target genes fibromodulin and bone sialoprotein in vivo and that it directly associates with these cofactors through protein-protein interactions. Phosphorylation of Osterix at Ser-73/77 increased its ability to recruit p300 and SWI/SNF to either fibromodulin or bone sialoprotein promoters. We therefore propose that Osterix binds to Sp1 sequences on target gene promoters and that its phosphorylation by p38 enhances recruitment of coactivators to form transcriptionally active complexes.

BMP-2 induces Osterix expression through up-regulation of Dlx5 and its phosphorylation by p38.

Osterix, a zinc-finger transcription factor, is specifically expressed in osteoblasts and osteocytes of all developing bones. Because no bone formation occurs in Osterix null mice, Osterix is thought to be an essential regulator of osteoblast differentiation. We report that bone morphogenetic protein-2 (BMP-2) induces an increase in Osterix expression, which is mediated through a homeodomain sequence located in the proximal region of the Osterix promoter. Our results demonstrate that induction of Dlx5 by BMP-2 mediates Osterix transcriptional activation. First, BMP-2 induction of Dlx5 precedes the induction of Osterix. Second, Dlx5 binds to the BMP-responsive homeodomain sequences both in vitro and in vivo. Third, Dlx5 overexpression and knock-down assays demonstrate its role in activating Osterix expression in response to BMP-2. Furthermore, we show that Dlx5 is a novel substrate for p38 MAPK in vitro and in vivo and that Ser-34 and Ser-217 are the sites phosphorylated by p38. Phosphorylation at Ser-34/217 increases the transactivation potential of Dlx5. Thus, we propose that BMP activates expression of Osterix through the induction of Dlx5 and its further transcriptional activation by p38-mediated phosphorylation.