Activin Receptor Type 2A (ACVR2A) Functions Directly in Osteoblasts as a Negative Regulator of Bone Mass

Bone and skeletal muscle mass are highly correlated in mammals, suggesting the existence of common anabolic signaling networks that coordinate the development of these two anatomically adjacent tissues. The activin signaling pathway is an attractive candidate to fulfill such a role. Here, we generated mice with conditional deletion of activin receptor (ACVR) type 2A, ACVR2B, or both, in osteoblasts, to determine the contribution of activin receptor signaling in regulating bone mass. Immunohistochemistry localized ACVR2A and ACVR2B to osteoblasts and osteocytes. Primary osteoblasts expressed activin signaling components, including ACVR2A, ACVR2B, and ACVR1B (ALK4) and demonstrated increased levels of phosphorylated Smad2/3 upon exposure to activin ligands. Osteoblasts lacking ACVR2B did not show significant changes in vitro. However, osteoblasts deficient in ACVR2A exhibited enhanced differentiation indicated by alkaline phosphatase activity, mineral deposition, and transcriptional expression of osterix, osteocalcin, and dentin matrix acidic phosphoprotein 1. To investigate activin signaling in osteoblasts in vivo, we analyzed the skeletal phenotypes of mice lacking these receptors in osteoblasts and osteocytes (osteocalcin-Cre). Similar to the lack of effect in vitro, ACVR2B-deficient mice demonstrated no significant change in any bone parameter. By contrast, mice lacking ACVR2A had significantly increased femoral trabecular bone volume at 6 weeks of age. Moreover, mutant mice lacking both ACVR2A and ACVR2B demonstrated sustained increases in trabecular bone volume, similar to those in ACVR2A single mutants, at 6 and 12 weeks of age. Taken together, these results indicate that activin receptor signaling, predominantly through ACVR2A, directly and negatively regulates bone mass in osteoblasts

Metastasis of hormone-independent breast cancer to lung and bone is decreased by α-difluoromethylornithine treatment

INTRODUCTION: Polyamines affect proliferation, differentiation, migration and apoptosis of cells, indicating their potential as a target for cancer chemotherapy. Ornithine decarboxylase converts ornithine to putrescine and is the rate-limiting step in polyamine synthesis. α-Difluoromethylornithine (DFMO) irreversibly inhibits ornithine decarboxylase and MDA-MB-435 human breast cancer metastasis to the lung without blocking orthotopic tumor growth. This study tested the effects of DFMO on orthotopic tumor growth and lung colonization of another breast cancer cell line (MDA-MB-231) and the effects on bone metastasis of MDA-MB-435 cells. METHODS: MDA-MB-231 cells were injected into the mammary fat pad of athymic mice. DFMO treatment (2% per orally) began at the day of tumor cell injection or 21 days post injection. Tumor growth was measured weekly. MDA-MB-231 cells were injected into the tail vein of athymic mice. DFMO treatment began 7 days prior to injection, or 7 or 14 days post injection. The number and incidence of lung metastases were determined. Green fluorescent protein-tagged MDA-MB-435 cells were injected into the left cardiac ventricle in order to assess the incidence and extent of metastasis to the femur. DFMO treatment began 7 days prior to injection. RESULTS: DFMO treatment delayed MDA-MB-231 orthotopic tumor growth to a greater extent than growth of MDA-MB-435 tumors. The most substantial effect on lung colonization by MDA-MB-231 cells occurred when DFMO treatment began 7 days before intravenous injection of tumor cells (incidence decreased 28% and number of metastases per lung decreased 35–40%). When DFMO treatment began 7 days post injection, the incidence and number of metastases decreased less than 10%. Surprisingly, treatment initiated 14 days after tumor cell inoculation resulted in a nearly 50% reduction in the number of lung metastases without diminishing the incidence. After intracardiac injection, DFMO treatment decreased the incidence of bone metastases (55% vs 87%) and the area occupied by the tumor (1.66 mm(2 )vs 4.51 mm(2), P < 0.05). CONCLUSION: Taken together, these data demonstrate that DFMO exerts an anti-metastatic effect in more than one hormone-independent breast cancer, for which no standard form of biologically-based treatment exists. Importantly, the data show that DFMO is effective against metastasis to multiple sites and that treatment is generally more effective when administered early

Memantine improves buprenorphine/naloxone treatment for opioid dependent young adults

BACKGROUND: Opioid use disorders are considered a serious public health problem among young adults. Current treatment is limited to long-term opioid substitution therapy, with high relapse rates after discontinuation. This study evaluated the co-administration of memantine to brief buprenorphine pharmacotherapy as a treatment alternative. METHODS: 13-week double-blind placebo-controlled trial evaluating 80 young adult opioid dependent participants treated with buprenorphine/naloxone 16-4mg/day and randomized to memantine (15mg or 30mg) or placebo. Primary outcomes were a change in the weekly mean proportion of opioid use, and cumulative abstinence rates after rapid buprenorphine discontinuation on week 9. RESULTS: Treatment retention was not significantly different between groups. The memantine 30mg group was significantly less likely to relapse and to use opioids after buprenorphine discontinuation. Among participants abstinent on week 8, those in the memantine 30mg group (81.9%) were significantly less likely to relapse after buprenorphine was discontinued compared to the placebo group (30%) (p \u3c 0.025). Also, the memantine 30mg group had significantly reduced opioid use (mean=0, SEM+/-0.00) compared to the placebo group (mean=0.33, SEM+/-0.35; p \u3c 0.004) during the last 2 weeks of study participation. CONCLUSIONS: Memantine 30mg significantly improved short-term treatment with buprenorphine/naloxone for opioid dependent young adults by reducing relapse and opioid use after buprenorphine discontinuation. Combined short-term treatment with buprenorphine/naloxone may be an effective alternative treatment to long-term methadone or buprenorphine maintenance in young adults

Deletion of IGF-I Receptor (IGF-IR) in Primary Osteoblasts Reduces GH-Induced STAT5 Signaling

GH promotes longitudinal growth and regulates multiple cellular functions in humans and animals. GH signals by binding to GH receptor (GHR) to activate the tyrosine kinase, Janus kinase 2 (JAK2), and downstream pathways including signal transducer and activator of transcription 5 (STAT5), thereby regulating expression of genes including IGF-I. GH exerts effects both directly and via IGF-I, which signals by activating the IGF-I receptor (IGF-IR). IGF-IR is a cell surface receptor that contains intrinsic tyrosine kinase activity within its intracellular domain. In this study, we examined the potential role of IGF-IR in facilitating GH-induced signal transduction, using mouse primary calvarial osteoblasts with Lox-P sites flanking both IGF-IR alleles. These cells respond to both GH and IGF-I and in vitro infection with an adenovirus that drives expression of Cre recombinase (Ad-Cre) dramatically reduces IGF-IR abundance without affecting the abundance of GHR, JAK2, STAT5, or ERK. Notably, infection with Ad-Cre, but not a control adenovirus, markedly inhibited acute GH-induced STAT5 activity (more than doubling the ED50 and reducing the maximum activity by nearly 50%), while sparing GH-induced ERK activity, and markedly inhibited GH-induced transactivation of a STAT5-dependent luciferase reporter. The effect of Ad-Cre on GH signaling was specific, as platelet-derived growth factor-induced signaling was unaffected by Ad-Cre-mediated reduction of IGF-IR. Ad-Cre-mediated inhibition of GH signaling was reversed by adenoviral reexpression of IGF-IR, but not by infection with an adenovirus that drives expression of a hemagglutination-tagged somatostatin receptor, which drives expression of the unrelated somatostatin receptor, and Ad-Cre infection of nonfloxed osteoblasts did not affect GH signaling. Notably, infection with an adenovirus encoding a C-terminally truncated IGF-IR that lacks the tyrosine kinase domain partially rescued both acute GH-induced STAT5 activity and GH-induced IGF-I gene expression in cells in which endogenous IGF-IR was reduced. These data, in concert with our earlier findings that GH induces a GHR-JAK2-IGF-IR complex, suggest a novel function for IGF-IR. In addition to its role as a key IGF-I signal transducer, this receptor may directly facilitate acute GH signaling. The implications of these findings are discussed

Α-Difluoromethylornithine (DFMO) treatment decreases lung metastasis of MDA-MB-231 cells

<p><b>Copyright information:</b></p><p>Taken from "Metastasis of hormone-independent breast cancer to lung and bone is decreased by α-difluoromethylornithine treatment"</p><p>Breast Cancer Research 2005;7(5):R819-R827.</p><p>Published online 9 Aug 2005</p><p>PMCID:PMC1242150.</p><p>Copyright © 2005 Richert et al, licensee BioMed Central Ltd.</p> MDA-MB-231 cells were injected into the tail vein of athymic mice. DFMO treatment (2% per orally) began either 7 days prior to (DFMO-7), or 7 days (DFMO+7) or 14 days (DMFO+14) after, tumor cell injection. Mice were killed either 4 weeks (Experiment 1) or 6 weeks post injection (Experiment 2). Lungs were removed and fixed in Bouin's fixative diluted 1:5 in neutral buffered formalin. Each lung was examined for the presence of surface metastases. The incidence of mice with lung metastases was graphed as a percentage of the total number of mice (= 10 for each group). The average number of metastases per lung was graphed as a percentage of the control (= 10 for each group). Experiment 1 (black) and Experiment 2 (small hatched) are graphed separately as well as combined (large hatched)

Α-Difluoromethylornithine (DFMO) (2% per orally in drinking water) decreases polyamine levels in mammary fat pad tumors of MDA-MB-231 cells

<p><b>Copyright information:</b></p><p>Taken from "Metastasis of hormone-independent breast cancer to lung and bone is decreased by α-difluoromethylornithine treatment"</p><p>Breast Cancer Research 2005;7(5):R819-R827.</p><p>Published online 9 Aug 2005</p><p>PMCID:PMC1242150.</p><p>Copyright © 2005 Richert et al, licensee BioMed Central Ltd.</p> Polyamine levels were determined in control subcutaneous (s.c.) and mammary fat pad (mfp) tumors as well as in DFMO-treated tumors grown in the mammary fat pad. DFMO treatment began at either 7 days prior to tumor cell injections (DFMO-7), on the day of injection (DFMO 0) or 21 days post injection (DFMO+21). Putrescine (black), spermidine (diagonal line) and spermine (cross-hatched) levels are graphed as nanomoles per milligram of tumor

Α-Difluoromethylornithine (DFMO) treatment affects both cell proliferation and cell death in MDA-MB-231 orthotopic tumors

<p><b>Copyright information:</b></p><p>Taken from "Metastasis of hormone-independent breast cancer to lung and bone is decreased by α-difluoromethylornithine treatment"</p><p>Breast Cancer Research 2005;7(5):R819-R827.</p><p>Published online 9 Aug 2005</p><p>PMCID:PMC1242150.</p><p>Copyright © 2005 Richert et al, licensee BioMed Central Ltd.</p> TUNEL analysis and BrdU analysis of MDA-MB-231 orthotopic tumors from either control mice or mice treated with 2% DFMO in the drinking water beginning at the day of injection

Α-Difluoromethylornithine (DFMO) decreases the incidence and size of bone metastases from MDA-MB-435 cells

<p><b>Copyright information:</b></p><p>Taken from "Metastasis of hormone-independent breast cancer to lung and bone is decreased by α-difluoromethylornithine treatment"</p><p>Breast Cancer Research 2005;7(5):R819-R827.</p><p>Published online 9 Aug 2005</p><p>PMCID:PMC1242150.</p><p>Copyright © 2005 Richert et al, licensee BioMed Central Ltd.</p> Green fluorescent protein-labeled MDA-MB-435 cells were injected intracardially into athymic mice. DFMO (2% per orally in drinking water ) treatment began 7 days prior to injection and continued for 6 weeks post injection. Bone metastases were visualized using a fluorescence microscope. Photographs of femurs from control (= 10) and DFMO-treated (= 10) mice show the presence and size of the bone metastases. Incidence of metastasis was determined by counting the number of mice with green fluorescence in the femur regardless of size of the mass. Image analysis was used to quantify fluorescence in each bone. The area of tumor in bone was quantified by comparing the total area of the femur with the area containing green fluorescence. The percentage of bone occupied by the tumor was also determined