Probes for INS

Cancer-associated fibroblasts (CAFs) play important roles in cancer progression through their complex interactions with cancer cells. The secreted bone morphogenetic protein antagonist, gremlin1 (GREM1) is expressed by the CAFs of basal cell carcinomas (BCCs), and promotes the growth of cancer cells. In this study, we investigated the expression of GREM1 mRNAs in various benign and malignant skin tumors, including various BCC subtypes. Analysis by RNA in situ hybridization (ISH) revealed that fibroblasts in the scar tissue expressed GREM1 and α-smooth muscle actin (α-SMA), whereas resident fibroblasts in the dermis of the normal skin did not express GREM1. Real-time polymerase chain reaction analysis showed significantly higher GREM1 expression in skin cancers and pilomatricomas (PMCs) than in other benign skin tumors. Tissue microarrays analyzed by RNA ISH for GREM1 expression also demonstrated that 23% of BCCs, 42% of squamous cell carcinomas, 20% of melanomas, and 90% of PMCs were positive for GREM1 expression, whereas trichoepitheliomas, eccrine poromas, hidradenomas, and spiradenomas were negative for GREM1 expression. Most BCCs that were GREM1 expression positive were of desmoplastic or mixed subtypes, and GREM1 expression was localized to activated myofibroblasts at the tumoral-stromal interface. Interestingly, most PMCs harbored GREM1-expressing fibroblasts, probably because of the inflammatory responses caused by foreign body reactions to keratin. Additionally, in BCCs, stromal GREM1 expression had a strong correlation with CD10 expression. In conclusion, GREM1 is frequently expressed by myofibroblasts in scars or in the stroma of basal cell carcinomas, suggesting that GREM1 expression can be a marker for activated myofibroblasts in the cancer stroma or in scar tissue.

Cancer associated fibroblasts (CAFs) are the dominant cell population in the cancer stroma. Gremlin 1 (GREM1), an antagonist of the bone morphogenetic protein pathway, is expressed by CAFs in a variety of human cancers. However, its biological significance for cancer patients is largely unknown. We applied RNA in situ hybridization (ISH) to evaluate the prognostic value of stromal GREM1 expression in a large cohort of 670 colorectal cancers (CRCs). Overall GREM1 expression in CRCs was lower than that of the matched normal mucosa, and GREM1 expression had a strong positive correlation with BMI1 and inverse correlations with EPHB2 and OLFM4. RNA ISH localized the GREM expression to smooth muscle cells of the muscularis mucosa, fibroblasts around crypt bases and in the submucosal space of a normal colon. In various colon polyps, epithelial GREM1 expression was exclusively observed in traditional serrated adenomas. In total, 44% of CRCs were positive for stromal GREM1, which was associated with decreased lymphovascular invasion, a lower cancer stage, and nuclear β-catenin staining. Stromal GREM1 was significantly associated with improved recurrence-free and overall survival, although it was not found to be an independent prognostic marker in multivariate analyses. In addition, for locally advanced stage II and III CRCs, it was associated with better, stage-independent clinical outcomes. In summary, CRCs are frequently accompanied by GERM1-expressing fibroblasts, which are closely associated with low lymphovascular invasion and a better prognosis, suggesting stromal GREM1 as a potential biomarker and possible candidate for targeted therapy in the treatment of CRCs.

Deficiency of vascular endothelial growth factor A (VEGF) has been associated with severe craniofacial anomalies in both humans and mice. Cranial neural crest cell (NCC)-derived VEGF regulates proliferation, vascularization and ossification of cartilage and membranous bone. However, the function of VEGF derived from specific subpopulations of NCCs in controlling unique aspects of craniofacial morphogenesis is not clear. In this study a conditional knockdown strategy was used to genetically delete Vegfa expression in Osterix (Osx) and collagen II (Col2)-expressing NCC descendants. No major defects in calvaria and mandibular morphogenesis were observed upon knockdown of VEGF in the Col2+ cell population. In contrast, loss of VEGF in Osx+ osteoblast progenitor cells led to reduced ossification of calvarial and mandibular bones without affecting the formation of cartilage templates in newborn mice. The early stages of ossification in the developing jaw revealed decreased initial mineralization levels and a reduced thickness of the collagen I (Col1)-positive bone template upon loss of VEGF in Osx+ precursors. Increased numbers of proliferating cells were detected within the jaw mesenchyme of mutant embryos. Explant culture assays revealed that mandibular osteogenesis occurred independently of paracrine VEGF action and vascular development. Reduced VEGF expression in mandibles coincided with increased phospho-Smad1/5 (P-Smad1/5) levels and bone morphogenetic protein 2 (Bmp2) expression in the jaw mesenchyme. We conclude that VEGF derived from Osx+ osteoblast progenitor cells is required for optimal ossification of developing mandibular bones and modulates mechanisms controlling BMP-dependent specification and expansion of the jaw mesenchyme.

Abstract

BACKGROUND/AIMS:

Murine nephrotoxic nephritis (NTN) is a well-established model resembling chronic kidney disease. Investigating gene expression patterns separately in the glomerular and cortical tubulointerstitial structure could provide new knowledge about structure-specific changes in expression of genes in the NTN model.

METHODS:

Glomerular, cortical tubulointerstitial and whole kidney tissues from mice subjected to nephrotoxic serum (NTS) or phosphate buffered saline (PBS) were collected on day 7, 21 and 42 using laser microdissection (LMD). Total RNA was extracted and subjected to nCounter NanoString. Histology, immunohistochemistry, in situ hybridization and/or quantitative real time PCR (qRT PCR) were performed to confirm regulation of selected genes.

RESULTS:

LMD provided detailed information about genes that were regulated differently between structures over time. Some of the fibrotic and inflammatory genes (Col1a1, Col3a1 and Ccl2) were upregulated in both structures, whereas other genes such as Spp1 and Grem1 were differentially regulated suggesting spatial pathogenic mechanisms in the kidney. Downregulation of cortical tubulointerstitium genes involved in iron metabolism was detected along with iron accumulation.

CONCLUSION:

This study demonstrates several regulated genes in pathways important for the pathogenesis of the NTN model and that LMD identifies structure-specific changes in gene expression during disease development. Furthermore, this study shows the benefits of isolating glomeruli and cortical tubulointerstitium in order to identify gene regulation.

Microvascular endothelial cells (EC) display a high degree of phenotypic and functional heterogeneity among different organs. Organ-specific EC control their tissue microenvironment by angiocrine factors in health and disease. Liver sinusoidal EC (LSEC) are uniquely differentiated to fulfil important organ-specific functions in development, under homeostatic conditions, and in regeneration and liver pathology. Recently, Bmp2 has been identified by us as an organ-specific angiokine derived from LSEC. To study angiocrine Bmp2 signaling in the liver, we conditionally deleted Bmp2 in LSEC using EC subtype-specific Stab2-Cre mice. Genetic inactivation of hepatic angiocrine Bmp2 signaling in Stab2-Cre;Bmp2fl/fl(Bmp2LSECKO) mice caused massive iron overload in the liver, and increased serum iron levels and iron deposition in several organs similar to classic hereditary hemochromatosis. Iron overload was mediated by decreased hepatic expression of hepcidin, a key regulator of iron homeostasis. Thus, angiocrine Bmp2 signaling within the hepatic vascular niche represents a constitutive pathway indispensable for iron homeostasis in vivo that is non-redundant with Bmp6. Notably, we demonstrate that organ-specific angiocrine signaling is essential not only for the homeostasis of the respective organ, but also for the homeostasis of the whole organism.

Microvascular endothelial cells (ECs) display a high degree of phenotypic and functional heterogeneity among different organs. Organ-specific ECs control their tissue microenvironment by angiocrine factors in health and disease. Liver sinusoidal endothelial cells (LSECs) are uniquely differentiated to fulfill important organ-specific functions in development, under homeostatic conditions, and in regeneration and liver pathology. Recently, Bmp2 has been identified by us as an organ-specific angiokine derived from LSECs. To study angiocrine Bmp2 signaling in the liver, we conditionally deleted Bmp2 in LSECs using EC subtype-specific Stab2-Cre mice. Genetic inactivation of hepatic angiocrine Bmp2 signaling in Stab2-Cre;Bmp2fl/fl (Bmp2LSECKO) mice caused massive iron overload in the liver and increased serum iron levels and iron deposition in several organs similar to classic hereditary hemochromatosis. Iron overload was mediated by decreased hepatic expression of hepcidin, a key regulator of iron homeostasis. Thus, angiocrine Bmp2 signaling within the hepatic vascular niche represents a constitutive pathway indispensable for iron homeostasis in vivo that is nonredundant with Bmp6. Notably, we demonstrate that organ-specific angiocrine signaling is essential not only for the homeostasis of the respective organ but also for the homeostasis of the whole organism.

SHP2 is a ubiquitously expressed protein tyrosine phosphatase, which is involved in many signaling pathways to regulate the skeletal development. In endochondral ossification, SHP2 is known to modify the osteogenic fate of osteochondroprogenitors and to impair the osteoblastic transdifferentiation of hypertrophic chondrocytes. However, how SHP2 regulates osteoblast differentiation in intramembranous ossification remains incompletely understood. To address this question, we generated a mouse model to ablate SHP2 in the Prrx1-expressing mesenchymal progenitors by using "Cre-loxP"-mediated gene excision and examined the development of calvarial bone, in which the main process of bone formation is intramembranous ossification. Phenotypic characterization showed that SHP2 mutants have severe defects in calvarial bone formation. Cell lineage tracing and in situ hybridization data showed less osteoblast differentiation of mesenchymal cells and reduced osteogenic genes expression, respectively. Further mechanistic studies revealed enhanced TGFβ and suppressed BMP2 signaling in SHP2 ablated mesenchymal progenitors and their derivatives. Our study uncovered the critical role of SHP2 in osteoblast differentiation through intramembranous ossification and might provide a potential target to treat craniofacial skeleton disorders.

Grem1, an antagonist of bone morphogenetic proteins, plays a key role in embryogenesis. A highly specific temporospatial gradient of Grem1 and BMP signalling is critical to normal lung, kidney and limb development. Grem1 levels are increased in renal fibrotic conditions including acute kidney injury, diabetic nephropathy, chronic allograft nephropathy and immune glomerulonephritis. A small number of grem1-/- whole body knockout mice on a mixed genetic background (8 %) are viable, with a single, enlarged left kidney and grossly normal histology. Grem1-/- mice displayed mild renal dysfunction at 4 wk, which recovered by 16 wk. Tubular epithelial specific targeted deletion of Grem1 (Grem1-TEC-/-) mice displayed a milder response in both the acute injury and recovery phase of the folic acid model. Grem1-TEC-/- mice had smaller increases in indices of kidney damage compared to wild-type. In the recovery phase of the folic acid model, associated with renal fibrosis, Grem1-TEC-/- mice displayed reduced histological damage and an attenuated fibrotic gene response compared to wild-type controls. Together, these data demonstrated that Grem1 expression in the tubular epithelial compartment plays a significant role in the fibrotic response to renal injury in vivo.