A nuclear factor 1 binding site mediates the transcriptional activation of a type I collagen promoter by transforming growth factor-beta

Transforming growth factor-beta (TGF-beta) increases the steady-state RNA levels of several fibroblast extracellular matrix proteins. Using DNA transfection, we show that TGF-beta stimulates the activity of the mouse alpha 2(l) collagen promoter 5- to 10-fold in mouse NIH 3T3 and rat osteosarcoma cells. Deletion analysis indicates that a segment of this promoter between -350 and -300, overlapping a nuclear factor 1 (NF1) binding site, is needed for TGF-beta stimulation. A 3 bp substitution mutation abolishing NF1 binding to this site inhibits TGF-beta activation. Insertion of this NF1 binding site 5' to the SV40 early promoter makes the promoter TGF-beta inducible, but the 3 bp substitution does not. Similarly, when the NF1 binding site at the replication origin of adenovirus 2 and 5 is inserted 5' to the SV40 promoter, the promoter responds to TGF-beta. Therefore an NF1 binding site mediates the transcriptional activation of the mouse alpha 2(l) collagen promoter by TGF-beta

The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice

Nearly every extracellular ligand that has been found to play a role in regulating bone biology acts, at least in part, through MAPK pathways. Nevertheless, much remains to be learned about the contribution of MAPKs to osteoblast biology in vivo. Here we report that the p38 MAPK pathway is required for normal skeletogenesis in mice, as mice with deletion of any of the MAPK pathway member–encoding genes MAPK kinase 3 (Mkk3), Mkk6, p38a, or p38b displayed profoundly reduced bone mass secondary to defective osteoblast differentiation. Among the MAPK kinase kinase (MAP3K) family, we identified TGF-β–activated kinase 1 (TAK1; also known as MAP3K7) as the critical activator upstream of p38 in osteoblasts. Osteoblast-specific deletion of Tak1 resulted in clavicular hypoplasia and delayed fontanelle fusion, a phenotype similar to the cleidocranial dysplasia observed in humans haploinsufficient for the transcription factor runt-related transcription factor 2 (Runx2). Mechanistic analysis revealed that the TAK1–MKK3/6–p38 MAPK axis phosphorylated Runx2, promoting its association with the coactivator CREB-binding protein (CBP), which was required to regulate osteoblast genetic programs. These findings reveal an in vivo function for p38β and establish that MAPK signaling is essential for bone formation in vivo. These results also suggest that selective p38β agonists may represent attractive therapeutic agents to prevent bone loss associated with osteoporosis and aging

GSK-3β Function in Bone Regulates Skeletal Development, Whole-Body Metabolism, and Male Life Span

Glycogen synthase kinase 3 β (GSK-3β) is an essential negative regulator or “brake” on many anabolic-signaling pathways including Wnt and insulin. Global deletion of GSK-3β results in perinatal lethality and various skeletal defects. The goal of our research was to determine GSK-3β cell-autonomous effects and postnatal roles in the skeleton. We used the 3.6-kb Col1a1 promoter to inactivate the Gsk3b gene (Col1a1-Gsk3b knockout) in skeletal cells. Mutant mice exhibit decreased body fat and postnatal bone growth, as well as delayed development of several skeletal elements. Surprisingly, the mutant mice display decreased circulating glucose and insulin levels despite normal expression of GSK-3β in metabolic tissues. We showed that these effects are due to an increase in global insulin sensitivity. Most of the male mutant mice died after weaning. Prior to death, blood glucose changed from low to high, suggesting a possible switch from insulin sensitivity to resistance. These male mice die with extremely large bladders that are preceded by damage to the urogenital tract, defects that are also seen type 2 diabetes. Our data suggest that skeletal-specific deletion of GSK-3β affects global metabolism and sensitizes male mice to developing type 2 diabetes. (Endocrinology 154: 3702–3718, 2013

Recommandations pour l’utilisation de la toxine botulinique de type A (Botox®) dans l’hyperactivité vésicale réfractaire idiopathique

RésuméObjectifsDéfinir des recommandations pour l’utilisation pratique de la toxine botulinique de type A (BoNTA) dans l’hyperactivité vésicale réfractaire idiopathique (HAVRI).MéthodeÉlaboration de recommandations de bonne pratique par consensus formalisé, validées par un groupe de 13 experts puis par un groupe de lecture indépendant.RésultatsEn cas d’infection urinaire celle-ci doit être traitée et l’injection reportée. Avant l’injection, il est recommandé de s’assurer de la faisabilité et de l’acceptabilité de l’auto-sondage. L’injection peut être réalisée après une anesthésie locale urétro-vésicale (lidocaïne), éventuellement complétée par l’inhalation de protoxyde d’azote et parfois sous anesthésie générale. L’injection sera réalisée au bloc opératoire ou en salle d’endoscopie. La vessie ne doit pas être trop remplie (risque de perforation). Le traitement doit être appliqué en 10 à 20 injections de 0,5 à 1mL réparties de manière homogène dans la vessie en restant à distance des méats urétéraux. Il n’est pas recommandé de laisser en place une sonde vésicale sauf en cas d’hématurie importante. Le patient doit être surveillé jusqu’à la reprise mictionnelle. Une note d’information sur les effets indésirables éventuels doit lui être remise à sa sortie. Une consultation doit être prévue 3 mois après la première injection (calendrier mictionnel, débitmétrie, résidu post-mictionnel et examen cytobactériologique des urines). Un résidu >200mL et/ou symptomatique doit faire discuter des auto-sondages. Une nouvelle injection pourra être envisagée lorsque le bénéfice clinique de la précédente s’estompe (entre 6 et 9 mois).ConclusionsLe respect de ces recommandations devrait permettre une utilisation optimale de la BoNTA.Niveau de preuve3.SummaryObjectivesProvide guidelines for practical usage of botulinum toxin type A (BoNTA) for refractory idiopathic Overactive Bladder management.Patients and methodsGuidelines using formalized consensus guidelines method. These guidelines have been validated by a group of 13 experts quoting proposals, subsequently reviewed by an independent group of experts.ResultsIn the case of patients with urinary tract infection, it must be treated and injection postponed. Before proposing an injection, it is recommended to ensure the feasibility and acceptability of self-catheterisation by patient. The injection can be performed after local anesthesia of the bladder and urethra (lidocaine), supplemented where necessary by nitrous oxide inhalation and sometimes under general anesthesia. Injection is performed in the operating room or endoscopy suite. The bladder should not be too filled (increased risk of perforation). Treatment should be applied in 10 to 20 injections of 0.5 to 1mL homogeneously distributed in the bladder at a distance from the urethral orifices. It is not recommended to leave a urinary catheter in place except in cases of severe hematuria. The patient should be monitored until resumption of micturition. After the first injection, an appointment must be scheduled within 3 months (micturition diary, uroflowmetry, measurement of residual urine and urine culture). Performance of self-catheterisation should be questioned in the case of a symptomatic post-void residual and/or a residue>200mL. A new injection may be considered when the clinical benefit of the previous injection diminishes (between 6 and 9 months). A period of three months must elapse between each injection.ConclusionsImplementation of these guidelines may promote best practice usage of BoNTA with optimal risk/benefit ratio

The GENETPIG database: a tool for comparative mapping in pig (Sus scrofa)

The GENETPIG database has been established for storing and disseminating the results of the European project: ‘GENETPIG: identification of genes controlling economic traits in pig'. The partners of this project have mapped about 630 porcine and human ESTs onto the pig genome. The database collects the mapping results and links them to other sources of mapping data; this includes pig maps as well as available comparative mapping information. Functional annotation of the mapped ESTs is also given when a significant similarity to cognate genes was established. The database is accessible for consultation via the Internet at http://www.infobiogen.fr/services/Genetpi

Heterogeneity in reporting on urinary outcome and cure after surgical interventions for stress urinary incontinence in adult neuro-urological patients: A systematic review

Aims: To describe all outcome parameters and definitions of cure used to report on outcome of surgical interventions for stress urinary incontinence (SUI) in neuro-urological (NU) patients. Methods: This systematic review was performed and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. The study protocol was registered and published (CRD42016033303; http://www.crd.york.ac.uk/PROSPERO). Medline, Embase, Cochrane controlled trials databases, and clinicaltrial.gov were systematically searched for relevant publications until February 2017. Result

Human chondrogenic paraxial mesoderm, directed specification and prospective isolation from pluripotent stem cells

Directed specification and prospective isolation of chondrogenic paraxial mesoderm progeny from human pluripotent stem (PS) cells have not yet been achieved. Here we report the successful generation of KDR−PDGFRα+ progeny expressing paraxial mesoderm genes and the mesendoderm reporter MIXL1-GFP in a chemically defined medium containing the canonical WNT signaling activator, BMP-inhibitor, and the Nodal/Activin/TGFβ signaling controller. Isolated (GFP+)KDR−PDGFRα+ mesoderm cells were sensitive to sequential addition of the three chondrogenic factors PDGF, TGFβ and BMP. Under these conditions, the cells showed robust chondrogenic activity in micromass culture, and generated a hyaline-like translucent cartilage particle in serum-free medium. In contrast, both STRO1+ mesenchymal stem/stromal cells from adult human marrow and mesenchymal cells spontaneously arising from hPS cells showed a relatively weaker chondrogenic response in vitro, and formed more of the fibrotic cartilage particles. Thus, hPS cell-derived KDR−PDGFRα+ paraxial mesoderm-like cells have potential in engineered cartilage formation and cartilage repair

Molecular changes in articular cartilage and subchondral bone in the rat anterior cruciate ligament transection and meniscectomized models of osteoarthritis

<p>Abstract</p> <p>Background</p> <p>Osteoarthritis (OA) is a debilitating, progressive joint disease.</p> <p>Methods</p> <p>Similar to the disease progression in humans, sequential events of early cartilage degradation, subchondral osteopenia followed by sclerosis, and late osteophyte formation were demonstrated in the anterior cruciate ligament transection (ACLT) or ACLT with partial medial meniscectomy (ACLT + MMx) rat OA models. We describe a reliable and consistent method to examine the time dependent changes in the gene expression profiles in articular cartilage and subchondral bone.</p> <p>Results</p> <p>Local regulation of matrix degradation markers was demonstrated by a significant increase in mRNA levels of aggrecanase-1 and MMP-13 as early as the first week post-surgery, and expression remained elevated throughout the 10 week study. Immunohistochemistry confirmed MMP-13 expression in differentiated chondrocytes and synovial fibroblasts at week-2 and cells within osteophytes at week-10 in the surgically-modified-joints. Concomitant increases in chondrocyte differentiation markers, Col IIA and Sox 9, and vascular invasion markers, VEGF and CD31, peaked around week-2 to -4, and returned to Sham levels at later time points in both models. Indeed, VEGF-positive cells were found in the deep articular chondrocytes adjacent to subchondral bone. Osteoclastic bone resorption markers, cathepsin K and TRAP, were also elevated at week-2. Confirming bone resorption is an early local event in OA progression, cathepsin K positive osteoclasts were found invading the articular cartilage from the subchondral region at week 2. This was followed by late disease events, including subchondral sclerosis and osteophyte formation, as demonstrated by the upregulation of the osteoanabolic markers runx2 and osterix, toward week-4 to 6 post-surgery.</p> <p>Conclusions</p> <p>In summary, this study demonstrated the temporal and cohesive gene expression changes in articular cartilage and subchondral bone using known markers of OA progression. The findings here support genome-wide profiling efforts to elucidate the sequential and complex regulation of the disease.</p

BRAF Mutations in Advanced Cancers: Clinical Characteristics and Outcomes

BACKGROUND: Oncogenic BRAF mutations have been found in diverse malignancies and activate RAF/MEK/ERK signaling, a critical pathway of tumorigenesis. We examined the clinical characteristics and outcomes of patients with mutant (mut) BRAF advanced cancer referred to phase 1 clinic. METHODS: We reviewed the records of 80 consecutive patients with mutBRAF advanced malignancies and 149 with wild-type (wt) BRAF (matched by tumor type) referred to the Clinical Center for Targeted Therapy and analyzed their outcome. RESULTS: Of 80 patients with mutBRAF advanced cancer, 56 had melanoma, 10 colorectal, 11 papillary thyroid, 2 ovarian and 1 esophageal cancer. Mutations in codon 600 were found in 77 patients (62, V600E; 13, V600K; 1, V600R; 1, unreported). Multivariate analysis showed less soft tissue (Odds ratio (OR) = 0.39, 95%CI: 0.20-0.77, P = 0.007), lung (OR = 0.38, 95%CI: 0.19-0.73, p = 0.004) and retroperitoneal metastases (OR = 0.34, 95%CI: 0.13-0.86, p = 0.024) and more brain metastases (OR = 2.05, 95%CI: 1.02-4.11, P = 0.043) in patients with mutBRAF versus wtBRAF. Comparing to the corresponding wtBRAF, mutBRAF melanoma patients had insignificant trend to longer median survival from diagnosis (131 vs. 78 months, p = 0.14), while mutBRAF colorectal cancer patients had an insignificant trend to shorter median survival from diagnosis (48 vs. 53 months, p = 0.22). In melanoma, V600K mutations in comparison to other BRAF mutations were associated with more frequent brain (75% vs. 36.3%, p = 0.02) and lung metastases (91.6% vs. 47.7%, p = 0.007), and shorter time from diagnosis to metastasis and to death (19 vs. 53 months, p = 0.046 and 78 vs. 322 months, p = 0.024 respectively). Treatment with RAF/MEK targeting agents (Hazard ratio (HR) = 0.16, 95%CI: 0.03-0.89, p = 0.037) and any decrease in tumor size after referral (HR = 0.07, 95%CI: 0.015-0.35, p = 0.001) correlated with longer survival in mutBRAF patients. CONCLUSIONS: BRAF appears to be a druggable mutation that also defines subgroups of patients with phenotypic overlap, albeit with differences that correlate with histology or site of mutation