Jerez, Sofía

Liver macrophages are central in maintaining hepatic homeostasis and mediating immune responses during liver injury, including fibrosis. Macrophages may have proinflammatory or antiinflammatory properties, but which properties influence fibrosis remains unclear. To explore the role of macrophages in liver fibrosis, we performed single-cell RNA-seq in a mouse model of liver injury and found that macrophage diversity was increased. Marco was among the most significantly upregulated genes, and a population of Marco hi macrophages increased with injury and spatially segregated to nonfibrotic areas. The macrophage receptor with collagenous structure (MARCO) protein is a scavenger receptor expressed by specific subsets of macrophages, and its role in liver fibrosis is unclear. In vitro induction of Marco in bone marrow–derived macrophages decreased proinflammatory gene expression, increased antiinflammatory and antifibrotic gene expression, and enhanced phagocytosis, indicating a restorative phenotype. Adoptive transfer of MARCO + macrophages in a mouse model of liver fibrosis reduced the expression of extracellular matrix–associated (ECM-associated) genes in hepatic stellate cells (HSCs) and reduced collagen deposition, which did not occur with the transfer of MARCO – macrophages. Therefore, MARCO + macrophages have a tissue restorative role in the liver and attenuate fibrogenesis through interaction with HSCs, thereby providing a potential therapeutic pathway for liver fibrosis.

Portal hypertension (PHTN) is a severe complication of liver cirrhosis and is associated with intrahepatic sinusoidal remodeling induced by sinusoidal resistance and angiogenesis. Collagen type IV (COL4), a major component of basement membrane, forms in liver sinusoids upon chronic liver injury. However, the role, cellular source, and expression regulation of COL4 in liver diseases are unknown. Here, we examined how COL4 is produced and how it regulates sinusoidal remodeling in fibrosis and PHTN. Human cirrhotic liver sample RNA sequencing showed increased COL4 expression, which was further verified via immunofluorescence staining. Single-cell RNA sequencing identified liver sinusoidal endothelial cells (LSECs) as the predominant source of COL4 upregulation in mouse fibrotic liver. In addition, COL4 was upregulated in a TNF-α/NF-κB–dependent manner through an epigenetic mechanism in LSECs in vitro. Indeed, by utilizing a CRISPRi-dCas9-KRAB epigenome-editing approach, epigenetic repression of the enhancer-promoter interaction showed silencing of COL4 gene expression. LSEC-specific COL4 gene mutation or repression in vivo abrogated sinusoidal resistance and angiogenesis, which thereby alleviated sinusoidal remodeling and PHTN. Our findings reveal that LSECs promote sinusoidal remodeling and PHTN during liver fibrosis through COL4 deposition.

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.