Galindo, Mario

Both genomic and non‐genomic mechanisms control the acquisition of specialized cellular phenotypes during development. When differentiated cells enter and exit mitosis, chromosomal condensation prevents gene expression. Post‐mitotic re‐activation of transcription is controlled in part by epigenetic marks on DNA and histones that control chromatin accessibility. However, mRNA molecules also appear to be transferred to progeny cells by either symmetrical or asymmetrical transmission. We have shown that the mRNA for the bone‐related transcription factor Runx2 mRNAs is elevated in the G2 phase of the cell cycle, but not translated in osteoblasts. This observation suggests that Runx2 protein is translationally rather than transcriptionally controlled after mitosis to maintain the phenotypic identity of osteoblasts. We used RNA‐seq expression profiling to assess whether there are other gene regulatory factors that are transmitted through mitosis. Our main results show that mRNAs for transcription factors are typically depleted during mitosis. For example, while Runx2 mRNA is retained in mitotic cells, expression of the closely related genes Runx1 and Runx3 is reduced. Strikingly, expression of Osterix/Sp7 and MEF2C, two transcription factors known to cooperate biologically with Runx2 in promoting the osteoblastic state, is selectively retained in mitotic cells. Our data suggest that selective somatic transmission of mRNAs for transcription factors in osteoblasts may be a general mechanism by which cells maintain their identity during cell division in skeletal development. Support or Funding Information NIH R01 AR049069 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Abstract Osteosarcomas are bone tumors that frequently metastasize to the lung. Aberrant expression of the transcription factor, runt‐related transcription factor 2 (RUNX2), is a key pathological feature in osteosarcoma and associated with loss of p53 and miR‐34 expression. Elevated RUNX2 may transcriptionally activate genes mediating tumor progression and metastasis, including the RUNX2 target gene osteopontin ( OPN/SPP1 ). This gene encodes a secreted matricellular protein produced by osteoblasts to regulate bone matrix remodeling and tissue calcification. Here we investigated whether and how the RUNX2/OPN axis regulates lung metastasis of osteosarcoma. Importantly, RUNX2 depletion attenuates lung metastasis of osteosarcoma cells in vivo. Using next‐generation RNA‐sequencing, protein‐based assays, as well as the loss‐ and gain‐of‐function approaches in selected osteosarcoma cell lines, we show that osteopontin messenger RNA levels closely correlate with RUNX2 expression and that RUNX2 controls the levels of secreted osteopontin. Elevated osteopontin levels promote heterotypic cell–cell adhesion of osteosarcoma cells to human pulmonary microvascular endothelial cells, but not in the presence of neutralizing antibodies. Collectively, these findings indicate that the RUNX2/OPN axis regulates the ability of osteosarcoma cells to attach to pulmonary endothelial cells as a key step in metastasis of osteosarcoma cells to the lung.

AbstractOsteoblast differentiation is controlled by transcription factor RUNX2 which temporally activates or represses several bone‐related genes, including those encoding extracellular matrix proteins or factors that control cell‐cell, and cell‐matrix interactions. Cell‐cell communication in the many skeletal pericellular micro‐niches is critical for bone development and involves paracrine secretion of growth factors and morphogens. This paracrine signaling is in part regulated by “A Disintegrin And Metalloproteinase” (ADAM) proteins. These cell membrane‐associated metalloproteinases support proteolytic release (“shedding”) of protein ectodomains residing at the cell surface. We analyzed microarray and RNA‐sequencing data for Adam genes and show that Adam17, Adam10, and Adam9 are stimulated during BMP2 mediated induction of osteogenic differentiation and are robustly expressed in human osteoblastic cells. ADAM17, which was initially identified as a tumor necrosis factor alpha (TNFα) converting enzyme also called (TACE), regulates TNFα‐signaling pathway, which inhibits osteoblast differentiation. We demonstrate that Adam17 expression is suppressed by RUNX2 during osteoblast differentiation through the proximal Adam17 promoter region (−0.4 kb) containing two functional RUNX2 binding motifs. Adam17 downregulation during osteoblast differentiation is paralleled by increased RUNX2 expression, cytoplasmic‐nuclear translocation and enhanced binding to the Adam17 proximal promoter. Forced expression of Adam17 reduces Runx2 and Alpl expression, indicating that Adam17 may negatively modulate osteoblast differentiation. These findings suggest a novel regulatory mechanism involving a reciprocal Runx2‐Adam17 negative feedback loop to regulate progression through osteoblast differentiation. Our results suggest that RUNX2 may control paracrine signaling through regulation of ectodomain shedding at the cell surface of osteoblasts by directly suppressing Adam17 expression.

ABSTRACTOsteosarcoma is the most common malignant bone tumor in children and adolescents. Metastasis and poor responsiveness to chemotherapy in osteosarcoma correlates with over‐expression of the runt‐related transcription factor RUNX2, which normally plays a key role in osteogenic lineage commitment, osteoblast differentiation, and bone formation. Furthermore, WNT/β‐catenin signaling is over‐activated in osteosarcoma and promotes tumor progression. Importantly, the WNT/β‐catenin pathway normally activates RUNX2 gene expression during osteogenic lineage commitment. Therefore, we examined whether the WNT/β‐catenin pathway controls the tumor‐related elevation of RUNX2 expression in osteosarcoma. We analyzed protein levels and nuclear localization of β‐catenin and RUNX2 in a panel of human osteosarcoma cell lines (SAOS, MG63, U2OS, HOS, G292, and 143B). In all six cell lines, β‐catenin and RUNX2 are expressed to different degrees and localized in the nucleus and/or cytoplasm. SAOS cells have the highest levels of RUNX2 protein that is localized in the nucleus, while MG63 cells have the lowest RUNX2 levels which is mostly localized in the cytoplasm. Levels of β‐catenin and RUNX2 protein are enhanced in HOS, G292, and 143B cells after treatment with the GSK3β inhibitor SB216763. Furthermore, small interfering RNA (siRNA)‐mediated depletion of β‐catenin inhibits RUNX2 expression in G292 cells. Thus, WNT/β‐catenin activation is required for RUNX2 expression in at least some osteosarcoma cell types, where RUNX2 is known to promote expression of metastasis related genes. J. Cell. Biochem. 118: 3662–3674, 2017. © 2017 Wiley Periodicals, Inc.

ABSTRACTOsteogenic lineage commitment and progression is controlled by multiple signaling pathways (e.g., WNT, BMP, FGF) that converge on bone‐related transcription factors. Access of osteogenic transcription factors to chromatin is controlled by epigenetic regulators that generate post‐translational modifications of histones (“histone code”), as well as read, edit and/or erase these modifications. Our understanding of the biological role of epigenetic regulators in osteoblast differentiation remains limited. Therefore, we performed next‐generation RNA sequencing (RNA‐seq) and established which chromatin‐related proteins are robustly expressed in mouse bone tissues (e.g., fracture callus, calvarial bone). These studies also revealed that cells with increased osteogenic potential have higher levels of the H4K20 methyl transferase Suv420h2 compared to other methyl transferases (e.g., Suv39h1, Suv39h2, Suv420h1, Ezh1, Ezh2). We find that all six epigenetic regulators are transiently expressed at different stages of osteoblast differentiation in culture, with maximal mRNAs levels of Suv39h1 and Suv39h2 (at day 3) preceding maximal expression of Suv420h1 and Suv420h2 (at day 7) and developmental stages that reflect, respectively, early and later collagen matrix deposition. Loss of function analysis of Suv420h2 by siRNA depletion shows loss of H4K20 methylation and decreased expression of bone biomarkers (e.g., alkaline phosphatase/Alpl) and osteogenic transcription factors (e.g., Sp7/Osterix). Furthermore, Suv420h2 is required for matrix mineralization during osteoblast differentiation. We conclude that Suv420h2 controls the H4K20 methylome of osteoblasts and is critical for normal progression of osteoblastogenesis. J. Cell. Biochem. 118: 1262–1272, 2017. © 2016 Wiley Periodicals, Inc.

Epigenetic mechanisms mediate the acquisition of specialized cellular phenotypes during tissue development, maintenance and repair. When phenotype‐committed cells transit through mitosis, chromosomal condensation counteracts epigenetic activation of gene expression. Subsequent post‐mitotic re‐activation of transcription depends on epigenetic DNA and histone modifications, as well as other architecturally bound proteins that “bookmark” the genome. Osteogenic lineage commitment, differentiation and progenitor proliferation require the bone‐related runt‐related transcription factor Runx2. Here, we characterized a non‐genomic mRNA mediated mechanism by which osteoblast precursors retain their phenotype during self‐renewal. We show that osteoblasts produce maximal levels of Runx2 mRNA, but not protein, prior to mitotic cell division. Runx2 mRNA partitions symmetrically between daughter cells in a non‐chromosomal tubulin‐containing compartment. Subsequently, transcription‐independent de novo synthesis of Runx2 protein in early G1 phase results in increased functional interactions of Runx2 with a representative osteoblast‐specific target gene (osteocalcin/BGLAP2) in chromatin. Somatic transmission of Runx2 mRNAs in osteoblasts and osteosarcoma cells represents a versatile mechanism for translational rather than transcriptional induction of this principal gene regulator to maintain osteoblast phenotype identity after mitosis. J. Cell. Physiol. 231: 1001–1014, 2016. © 2015 Wiley Periodicals, Inc.