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

IKKα promotes lung adenocarcinoma growth through ERK signaling activation via DARPP-32-mediated inhibition of PP1 activity

Abstract Non-small cell lung cancer (NSCLC) accounts for 80–85% cases of lung cancer cases. Diagnosis at advanced stages is common, after which therapy-refractory disease progression frequently occurs. Therefore, a better understanding of the molecular mechanisms that control NSCLC progression is necessary to develop new therapies. Overexpression of IκB kinase α (IKKα) in NSCLC correlates with poor patient survival. IKKα is an NF-κB-activating kinase that is important in cell survival and differentiation, but its regulation of oncogenic signaling is not well understood. We recently demonstrated that IKKα promotes NSCLC cell migration by physically interacting with dopamine- and cyclic AMP-regulated phosphoprotein, Mr 32000 (DARPP-32), and its truncated splice variant, t-DARPP. Here, we show that IKKα phosphorylates DARPP-32 at threonine 34, resulting in DARPP-32-mediated inhibition of protein phosphatase 1 (PP1), subsequent inhibition of PP1-mediated dephosphorylation of ERK, and activation of ERK signaling to promote lung oncogenesis. Correspondingly, IKKα ablation in human lung adenocarcinoma cells reduced their anchorage-independent growth in soft agar. Mice challenged with IKKα-ablated HCC827 cells exhibited less lung tumor growth than mice orthotopically administered control HCC827 cells. Our findings suggest that IKKα drives NSCLC growth through the activation of ERK signaling via DARPP-32-mediated inhibition of PP1 activity

Protein kinase D up-regulates transcription of VEGF receptor-2 in endothelial cells by suppressing nuclear localization of the transcription factor AP2β

Vascular endothelial growth factor A (VEGF) signals primarily through its cognate receptor VEGF receptor-2 (VEGFR-2) to control vasculogenesis and angiogenesis, key physiological processes in cardiovascular disease and cancer. In human umbilical vein endothelial cells (HUVECs), knockdown of protein kinase D-1 (PKD1) or PKD2 down-regulates VEGFR-2 expression and inhibits VEGF-induced cell proliferation and migration. However, how PKD regulates VEGF signaling is unclear. Previous bioinformatics analyses have identified binding sites for the transcription factor activating enhancer-binding protein 2 (AP2) in the VEGFR-2 promoter. Using ChIP analyses, here we found that PKD knockdown in HUVECs increases binding of AP2β to the VEGFR-2 promoter. Luciferase reporter assays with serial deletions of AP2-binding sites within the VEGFR-2 promoter revealed that its transcriptional activity negatively correlates with the number of these sites. Next we demonstrated that AP2β up-regulation decreases VEGFR-2 expression and that loss of AP2β enhances VEGFR-2 expression in HUVECs. In vivo experiments confirmed increased VEGFR-2 immunostaining in the spinal cord of AP2β knockout mouse embryos. Mechanistically, we observed that PKD phosphorylates AP2β at Ser258 and Ser277 and suppresses its nuclear accumulation. Inhibition of PKD activity with a pan-PKD inhibitor increased AP2β nuclear localization, and overexpression of both WT and constitutively active PKD1 or PKD2 reduced AP2β nuclear localization through a Ser258- and Ser277-dependent mechanism. Furthermore, substitution of Ser277 in AP2β increased its binding to the VEGFR-2 promoter. Our findings uncover evidence of a molecular pathway that regulates VEGFR-2 expression, insights that may shed light on the etiology of diseases associated with aberrant VEGF/VEGFR signaling.NHLBI, National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Heart Lung & Blood Institute (NHLBI) [HL140411]; NCI, National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Cancer Institute (NCI) [CA78383-20, CA187035, CA200572]; Florida Department of Health Cancer Research Chair Fund Florida Grant [3J-02]; American Heart AssociationAmerican Heart Association [13POST14510025, 19CDA34700013]; Mayo Clinic Ted and Loretta Rogers Cardiovascular Career Development Award Honoring Hugh C. SmithThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Lef1ΔN Binds β-Catenin and Increases Osteoblast Activity and Trabecular Bone Mass*

Lymphoid enhancer-binding factor (Lef) 1 is a high mobility group protein best known as a Wnt-responsive transcription factor that associates with β-catenin. Lef1ΔN is a short isoform of Lef1 that lacks the first 113 amino acids and a well characterized high affinity β-catenin binding domain present in the full-length protein. Both Lef1 isoforms bind DNA and regulate gene expression. We previously reported that Lef1 is expressed in proliferating osteoblasts and blocks osteocalcin expression. In contrast, Lef1ΔN is only detectable in the later stages of osteoblast differentiation and promotes osteogenesis in vitro. Here, we show that Lef1ΔN retains the ability to interact physically and functionally with β-catenin. Unlike what has been reported in T cells and colon cancer cell lines, Lef1ΔN activated gene transcription in the absence of exogenous β-catenin and cooperated with constitutively active β-catenin to stimulate gene transcription in mesenchymal and osteoblastic cells. Residues at the N terminus of Lef1ΔN were required for β-catenin binding and the expression of osteoblast differentiation genes. To determine the role of Lef1ΔN on bone formation in vivo, a Lef1ΔN transgene was expressed in committed osteoblasts using the 2.3-kb fragment of the type 1 collagen promoter. The Lef1ΔN transgenic mice had higher trabecular bone volume in the proximal tibias and L5 vertebrae. Histological analyses of tibial sections revealed no differences in osteoblast, osteoid, or osteoclast surface areas. However, bone formation and mineral apposition rates as well as osteocalcin levels were increased in Lef1ΔN transgenic mice. Together, our data indicate that Lef1ΔN binds β-catenin, stimulates Lef/Tcf reporter activity, and promotes terminal osteoblast differentiation

Neuropilin-2 promotes extravasation and metastasis by interacting with endothelial alpha 5 integrin

Metastasis, the leading cause of cancer death, requires tumor cell intravasation, migration through the bloodstream, arrest within capillaries, and extravasation to invade distant tissues. Few mechanistic details have been reported thus far regarding the extravasation process or re-entry of circulating tumor cells at metastatic sites. Here, we show that neuropilin-2 (NRP-2), a multifunctional nonkinase receptor for semaphorins, vascular endothelial growth factor (VEGF), and other growth factors, expressed on cancer cells interacts with α5 integrin on endothelial cells to mediate vascular extravasation and metastasis in zebrafish and murine xenograft models of clear cell renal cell carcinoma (RCC) and pancreatic adenocarcinoma. In tissue from patients with RCC, NRP-2 expression is positively correlated with tumor grade and is highest in metastatic tumors. In a prospectively acquired cohort of patients with pancreatic cancer, high NRP-2 expression cosegregated with poor prognosis. Through biochemical approaches as well as Atomic Force Microscopy (AFM), we describe a unique mechanism through which NRP-2 expressed on cancer cells interacts with α5 integrin on endothelial cells to mediate vascular adhesion and extravasation. Taken together, our studies reveal a clinically significant role of NRP-2 in cancer cell extravasation and promotion of metastasis

Creating clear and informative image-based figures for scientific publications

Scientists routinely use images to display data. Readers often examine figures first; therefore, it is important that figures are accessible to a broad audience. Many resources discuss fraudulent image manipulation and technical specifications for image acquisition; however, data on the legibility and interpretability of images are scarce. We systematically examined these factors in non-blot images published in the top 15 journals in 3 fields; plant sciences, cell biology, and physiology (n = 580 papers). Common problems included missing scale bars, misplaced or poorly marked insets, images or labels that were not accessible to colorblind readers, and insufficient explanations of colors, labels, annotations, or the species and tissue or object depicted in the image. Papers that met all good practice criteria examined for all image-based figures were uncommon (physiology 16%, cell biology 12%, plant sciences 2%). We present detailed descriptions and visual examples to help scientists avoid common pitfalls when publishing images. Our recommendations address image magnification, scale information, insets, annotation, and color and may encourage discussion about quality standards for bioimage publishing

Creating clear and informative image-based figures for scientific publications.

Scientists routinely use images to display data. Readers often examine figures first; therefore, it is important that figures are accessible to a broad audience. Many resources discuss fraudulent image manipulation and technical specifications for image acquisition; however, data on the legibility and interpretability of images are scarce. We systematically examined these factors in non-blot images published in the top 15 journals in 3 fields; plant sciences, cell biology, and physiology (n = 580 papers). Common problems included missing scale bars, misplaced or poorly marked insets, images or labels that were not accessible to colorblind readers, and insufficient explanations of colors, labels, annotations, or the species and tissue or object depicted in the image. Papers that met all good practice criteria examined for all image-based figures were uncommon (physiology 16%, cell biology 12%, plant sciences 2%). We present detailed descriptions and visual examples to help scientists avoid common pitfalls when publishing images. Our recommendations address image magnification, scale information, insets, annotation, and color and may encourage discussion about quality standards for bioimage publishing

Med8, Med18, and Med20 subunits of the Mediator head domain are interdependent upon each other for folding and complex formation

We have studied folding and complex formation of the yeast Mediator head-module protein subunits Med8, Med18, and Med20. Using a combination of immunoprecipitation, far-UV circular dichroism, and fluorescence measurements on recombinantly expressed and denatured proteins that were allowed to renature separately or in different combinations, we found that Med8, Med18, and Med20 can fold in different ways to form both soluble monomeric proteins and different distinct subcomplexes. However, the concurrent presence of all three protein subunits during the renaturation process is required for proper folding and trimer complex formation