Collagen 11a1 is Indirectly Activated by Lymphocyte Enhancer-Binding Factor 1 (Lef1) and Negatively Regulates Osteoblast Maturation

Alpha 1 (XI) collagen (Col11a1) is essential for normal skeletal development. Mutations in Col11a1 cause Marshall and Stickler syndromes, characterized by craniofacial abnormalities, nearsightedness and hearing abnormalities. Despite its link to human diseases, few studies have characterized the factors that control Col11a1 transcription. We previously identified Col11a1 as a differentially expressed gene in Lef1-suppressed MC3T3 preosteoblasts. Here we report that Lef1 activates the Col11a1 promoter. This activation is dependent upon the DNA binding domain of Lef1, but does not require the ß-catenin interaction domain, suggesting that it is not responsive to Wnt signals. Targeted deletion of Col11a1 with an antisense morpholino accelerated osteoblastic differentiation and mineralization in C2C12 cells, similar to what was observed in Lef1-suppressed MC3T3 cells. Moreover incubation with a purified Col11a1 N-terminal fragment, V1B, prevented alkaline phosphatase expression in MC3T3 and C2C12 cells. These results suggest that Lef1 is an activator of the Col11a1 promoter and that Col11a1 suppresses terminal osteoblast differentiation

Lymphocyte enhancer-binding factor 1 (Lef1) inhibits terminal differentiation of osteoblasts

Lef1 is a transcriptional regulator of the Wnt/beta-catenin signaling cascade. Wnts directly augment bone formation and osteoblast differentiation from mesenchymal stem cells by receptor-mediated pathways involving Lrp5 and Frizzled. We previously reported that Lef1 represses Runx2-dependent activation of the late osteoblast differentiation gene, osteocalcin. Lef1 is expressed in preosteoblasts but is undetectable in fully differentiated osteoblasts. To determine if downregulation of Lef1 is necessary for osteoblast maturation, we constitutively overexpressed Lef1 in MC3T3-E1 preosteoblasts. Lef1-overexpressing cells produced alkaline phosphatase (ALP) and osteocalcin later, and at lower levels than control cells. Moreover, the extracellular matrices of Lef1-overexpressing cell cultures never mineralized. To further examine the role of Lef1 in osteoblasts, we suppressed Lef1 expression in MC3T3-E1 cells by RNA interference. Transient expression of a Lef1 shRNA efficiently reduced murine Lef1 levels and transcriptional activity. Stable suppression of Lef1 in MC3T3 preosteoblasts did not affect proliferation or Runx2 levels; however, ALP production and matrix mineralization were accelerated by 3-4 days. Gene chip analyses identified 14 genes that are differentially regulated in Lef1-suppressed cells. These data outline a role for Lef1 in delaying osteoblast maturation and suggest that Lef1 controls the expression of multiple genes in osteoblasts

Geophysics Field Camp 2017

Electrical resistivity and induced polarization surveys were conducted in the Las Cienegas National Conservation Area for the Nature Conservancy and in accordance with the Bureau of Land Management. Data collected around Empire Ranch HQ shows a thin, high resistivity layer at the surface underlain by a lower resistivity layer, indicative of typical regional alluvium. H0 and H1 show resistivity of approximately 80 Ω-m down to 5 and 10 meters, underlain by 25 Ω-m and 20 Ω-m respectively. H2 and H3 shows resistivity of 60 Ω-m down to 5 and 15 meters, respectively, underlain by 20 Ω-m resistivity. Moving east, the data displays a distinct change. A thin, high-resistivity surface layer is underlain by an extremely low resistivity layer, typical of clays. H5, Z2 and H6 display surface resistivity between 20 Ω-m and 15 Ω-m, typical alluvium values. The massive underlying layer displays resistivity values of approximately 5 Ω-m. Continuing east, H7 shows a thick layer of high (80 Ω-m) resistivity surface material down to 25 meters, underlain again by a massive, low resistivity layer with a typical clay value of 5 Ω-m. H8 and Z3, collected in the Cieneguita cienegas complex, both display a 5 Ω-m surface layer down to 5 meters, indicative of clays. The surface layer is underlain by a 15 Ω-m, low-porosity layer, essentially devoid of clays, interpreted as Late Tertiary and Quaternary basin fill.The Geophysics Field Camp Reports are made available by the Laboratory for Advanced Subsurface Imaging (LASI) and the University of Arizona Libraries. Visit the LASI website for more information http://www.lasi.arizona.edu

Cell cycle related modulations in Runx2 protein levels are independent of lymphocyte enhancer-binding factor 1 (Lef1) in proliferating osteoblasts

Runt-related transcription factor Runx2 regulates osteogenic phenotype commitment and attenuates osteoblast growth. Runx2 levels are cell cycle regulated and maximal in the G1 phase of proliferating osteoblasts and during quiescence. The Wnt/Lrp5-Frizzled/beta-catenin/Lef-Tcf signaling cascade also controls progression along the osteogenic lineage with a net anabolic effect that promotes bone formation. However, Lef1 opposes the osteoblast maturation promoting activity of Runx2. Here we examined whether Lef1 controls Runx2 expression during the cell cycle or onset of quiescence in osteoblasts. We inhibited Lef1 expression using short hairpin (sh) RNA interference in stably transfected MC3T3-E1 cells. In asynchronously growing osteoblasts, expression of Lef1 shRNA diminishes Lef1 protein levels, but does not affect Runx2 levels. Cells arrested in different cell cycle stages using mimosine (late G1), hydroxyurea or aphidicolin (S phase) or nocodazole (mitosis) exhibit expected reductions in Runx2 protein levels despite reductions in Lef1. Serum deprived MC3T3-E1 cells normally upregulate Runx2 protein regardless of Lef1 deficiency, although loss of Lef1 reduces cyclin A and increases cyclin D1 expression upon serum withdrawal. Thus, Runx2 protein levels during the cell cycle and onset of quiescence are regulated independently of Lef1, one of the major transcriptional inducers of Wnt signaling in proliferating cells

Fine-Scale Population Genetic Structure and Dispersal of Juvenile Steelhead in the Bulkley-Morice River, British Columbia

A knowledge of fine-scale population genetic structure and patterns of dispersal is an essential component of any action to conserve genetic diversity and maintain population viability. We genotyped 417 juvenile steelhead Oncorhynchus mykiss from the main stem and tributaries of the Bulkley-Morice River, British Columbia, at 10 microsatellite loci to assess fine-scale population structure and the patterns and magnitude of juvenile dispersal and mixing. We detected significant genetic structuring among juvenile steelhead from seven tributaries of the Bulkley-Morice River (pairwise F-ST: 0.008-0.156) and found significant isolation by distance among the tributary populations (R-2 = 0.198, P = 0.038). These results reflect the homing behavior of spawning adults as well as the temporal stability of those populations. Genotype assignment of tributary-caught juveniles showed that rates of juvenile dispersal varied among tributaries. The assignment of juveniles sampled from the main stem of the river to source tributary populations suggested that long-distance movement in juvenile steelhead is common and that juveniles are well mixed in the main stem. Dispersal and fine-scale genetic structure in pristine steelhead populations are more complex than previously thought. Therefore, actions to conserve Bulkley-Morice River steelhead must strive to maintain the genetic diversity of tributary populations

Discovery of Small Molecule RIP1 Kinase Inhibitors for the Treatment of Pathologies Associated with Necroptosis

Potent inhibitors of RIP1 kinase from three distinct series, 1-aminoisoquinolines, pyrrolo­[2,3-b]­pyridines, and furo­[2,3-d]­pyrimidines, all of the type II class recognizing a DLG-out inactive conformation, were identified from screening of our in-house kinase focused sets. An exemplar from the furo­[2,3-d]­pyrimidine series showed a dose proportional response in protection from hypothermia in a mouse model of TNFα induced lethal shock