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Gremlin-1 is part of the TGF-β superfamily and is a BMP antagonist that blocks BMP signalling to precisely control BMP gradients. Gremlin-1 is primarily involved in organogenesis and limb patterning however, has recently been described as being involved in fibrotic diseases. Initially described as a key factor involved in diabetic kidney fibrosis due to being induced by high glucose, it has now been described as being associated with lung, liver, eye, and skin fibrosis. This suggests that it is a key conserved molecule mediating fibrotic events irrespective of organ. It appears that Gremlin-1 may have effects mediated by BMP-dependent and independent pathways. The aim of this review is to evaluate the role of Gremlin-1 in fibrosis, its mechanisms and if this can be targeted therapeutically in fibrotic diseases, which currently have very limited treatment options and are highly prevalent.

Members of the differential screening-selected gene aberrative in neuroblastoma (DAN) protein family are developmentally conserved extracellular binding proteins that antagonize bone morphogenetic protein (BMP) signaling. This protein family includes the Gremlin proteins, GREM1 and GREM2, which have key functions during embryogenesis and adult physiology. While BMPs play essential roles in ovarian follicle development, the role of the DAN family in female reproductive physiology is less understood. We generated mice null for Grem2 to determine its role in female reproduction in addition to screening patients with primary ovarian insufficiency (POI) for variants in GREM2. Grem2–/– mice are viable, but female Grem2–/– mice have diminished fecundity and irregular estrous cycles. This is accompanied by significantly reduced production of ovarian anti-Mullerian hormone (AMH) from small growing follicles, leading to a significant decrease in serum AMH. Surprisingly, as AMH is a well-established marker of the ovarian reserve, morphometric analysis of ovarian follicles showed maintenance of primordial follicles in Grem2–/– mice like wild-type (WT) littermates. While Grem2 mRNA transcripts were not detected in the pituitary, Grem2 is expressed in hypothalami of WT female mice, suggesting the potential for dysfunction in multiple tissues composing the hypothalamic–pituitary-ovarian axis that contribute to the subfertility phenotype. Additionally, screening 106 women with POI identified one individual with a heterozygous variant in GREM2 that lies within the predicted BMP-GREM2 interface. In total, these data suggest that Grem2 is necessary for female fecundity by playing a novel role in regulating the HPO axis and contributing to female reproductive disease. Summary sentence Female mice homozygous null for the BMP antagonist, Grem2, shows a complex reproductive phenotype, but which appears to primarily result from disruptions in the hypothalamic–pituitaryovarian axis leading to irregular estrous cycles. Graphical Abstract

International audience; OBJECTIVE: This study aimed to determine how different resistance training protocols affect gremlin 1, macrophage migration inhibitory factor (MIF), cardiometabolic, and anthropometric measures in obese men. METHODS: Forty-four males with obesity (weight: 93.2 ± 2.2 kg, BMI: 32.9 ± 1.2 kg/m(2), age: 27.5 ± 9.4 years) were randomly assigned to traditional resistance training (TRT, n = 11), circuit resistance training (CRT, n = 11), interval resistance training (IRT, n = 11) or control (C, n = 11) groups. TRT group performed ten exercises at 50% of 1RM with 14 repetitions for three sets and 30 seconds rest interval between exercises and 1.5 min rest between sets, the CRT protocol included three circuits of 10 exercises, at an intensity of 50% of 1-RM, 14 repetitions with a minimum rest (< 15 s) between exercises and 3 min rest between sets, and the IRT group performed two sets of the same exercises with 50% of 1 RM, and 14 repetitions were followed with active rest of 25% of 1RM and 14 repetitions. All resistance training groups performed 60 min per session resistance exercises, 3 days per week, for 12 weeks. Measurements were taken at baseline and after 12 weeks of exercise training. RESULTS: Resistance training (TRT, CRT, and IRT) significantly decreased plasma levels of gremlin (TRT from 231.0 ± 5.8 to 210.0 ± 11.6 ng/ml, CRT from 226.0 ± 7.6 to 188.0 ± 7.7 ng/ml and, IRT from 227.0 ± 6.3 to 183.0 ± 9.0 ng/ml, effect size (ES): 0.50), MIF (TRT from 251.0 ± 7.4 to 260.0 ± 6.5 ng/ml, CRT from 248.0 ± 10.9 to 214.0 ± 9.0 ng/ml and, IRT from 247.0 ± 8.9 to 196.0 ± 6.9 ng/ml, ES: 0.55) and CRP (TRT from 28.4 ± 1.7 to 23.3 ± 2.1 nmol/l, CRT from 28.5 ± 2.2 to 21.1 ± 1.8 nmol/l, IRT from 28.1 ± 1.3 to 20.8 ± 1.3 nmol/l, ES: 0.49) compared to the control group (p

In Glioblastoma (GBM) brain tumors, both Gremlin-1 and Noggin are reported to bind to BMP and inhibit BMP-signaling, thereby allowing the cell to maintain tumorous morphology. Enlisting the interfacial residues important for protein-protein complex formation between BMPs (BMP-2 and BMP-7) and antagonists (Gremlin-1 and Noggin), we analyzed the structural basis of their interactions. We found possible key mutations that destabilize these complexes, which may prevent GBM development. It was also observed that when the interfacial residues were either mutated to histidine or tryptophan, it led to higher destabilization energy values. Besides, our study of the Noggin interactive model of BMP-2 suggested preferential binding at binding site II over binding site I. In the case of Gremlin-1 and BMPs, our research, along with few previous studies, indicates a close-ended cis-trans interactive model.

Background Gremlin-1 (GREM1), one of the bone morphogenetic protein antagonists, is involved in organogenesis, tissue differentiation and kidney development. However, the role of GREM1 in cancer progression and its underlying mechanisms remain poorly understood. Methods The role of GREM1 in breast cancer progression was assessed by measuring cell viability, colony formation, 3D tumour spheroid formation/invasion and xenograft tumour formation. Chromatin immunoprecipitation, a luciferase reporter assay and flow cytometry were performed to investigate the molecular events in which GREM1 is involved. Results GREM1 expression was elevated in breast cancer cells and tissues obtained from breast cancer patients. Its overexpression was associated with poor prognosis in breast cancer patients, especially those with oestrogen receptor (ER)-negative tumours. GREM1 knockdown inhibited the proliferation of breast cancer cells and xenograft mammary tumour growth, while its overexpression enhanced their viability, growth and invasiveness. Oestrogen-related receptor α (ERRα), an orphan nuclear hormone receptor, directly interacted with the GREM1 promoter and increased the expression of GREM1. GREM1 also enhanced the promoter activity of ESRRA encoding ERRα, comprising a positive feedback loop. Notably, GREM1 bound to and activated EGFR, a well-known upstream regulator of ERRα. Conclusions Our study suggests that the GREM1–ERRα axis can serve as a potential therapeutic target in the management of cancer, especially ER-negative tumour.

The intestinal epithelium is perpetually renewed from a stem cell niche in the base of crypts to maintain a healthy bowel mucosa. Exit from this niche and maturation of epithelial cells requires tightly controlled gradients in BMP signalling, progressing from low BMP signalling at the crypt base to high signalling at the luminal surface. The BMP antagonist gremlin 1 (Grem1) is highly expressed by subepithelial myofibroblasts adjacent to the intestinal crypts but its role in regulating the stem cell niche and epithelial renewal in vivo has not been explored. To explore the effects of Grem1 loss in adulthood following normal growth and development, we bred mice (ROSA26CreER-Grem1 flx/flx ) in which Grem1 could be deleted by tamoxifen administration. While Grem1 remained intact, these mice were healthy, grew normally, and reproduced successfully. Following Grem1 depletion, the mice became unwell and were euthanised (at 7-13 days). Post-mortem examination revealed extensive mucosal abnormalities throughout the small and large intestines with failure of epithelial cell replication and maturation, villous atrophy, and features of malabsorption. Bone marrow hypoplasia was also observed with associated early haematopoietic failure. These results demonstrate an essential homeostatic role for gremlin 1 in maintaining normal bowel epithelial function in adulthood, suggesting that abnormalities in gremlin 1 expression can contribute to enteropathies. We also identified a previously unsuspected requirement for gremlin 1 in normal haematopoiesis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The bone morphogenetic protein (BMP) antagonist gremlin 1 plays a central role in the pathogenesis of hypoxic pulmonary hypertension (HPH). Recently, non-canonical functions of gremlin 1 have been identified, including specific binding to the vascular endothelial growth factor receptor-2 (VEGFR2). We tested the hypothesis that gremlin 1 modulates VEGFR2 signaling in the pulmonary microvascular endothelium. We examined the effect of gremlin 1 haploinsufficiency on the expression of VEGF responsive genes and proteins in the hypoxic (10% O(2)) murine lung in vivo. Using human microvascular endothelial cells in vitro we examined the effect of gremlin 1 on VEGF signaling. Gremlin 1 haploinsufficiency (Grem1(+/–)) attenuated the hypoxia-induced increase in gremlin 1 observed in the wild-type mouse lung. Reduced gremlin 1 expression in hypoxic Grem1(+/–) mice restored VEGFR2 expression and endothelial nitric oxide synthase (eNOS) expression and activity to normoxic values. Recombinant monomeric gremlin 1 inhibited VEGFA-induced VEGFR2 activation, downstream signaling, and VEGF-induced increases in Bcl-2, cell number, and the anti-apoptotic effect of VEGFA in vitro. These results show that the monomeric form of gremlin 1 acts as an antagonist of VEGFR2 activation in the pulmonary microvascular endothelium. Given the previous demonstration that inhibition of VEGFR2 causes marked worsening of HPH, our results suggest that increased gremlin 1 in the hypoxic lung, in addition to blocking BMP receptor type-2 (BMPR2) signaling, contributes importantly to the development of PH by a non-canonical VEGFR2 blocking activity.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β family of proteins that have been implicated in the paracrine regulation of granulosa cell (GC) function, but whether responses to BMPs change with follicular size or interact with connective tissue growth factor (CTGF) or BMP antagonists (e.g., gremlin [GREM]) to directly affect GC function of cattle is unknown. Therefore, to determine the effects of BMP4 on proliferation and steroidogenesis of GCs and its interaction with GREM or CTGF, experiments were conducted using bovine GC cultures. In vitro, BMP4 (30 ng/mL) inhibited (P < 0.05) follicle-stimulating hormone (FSH) plus insulin-like growth factor 1 (IGF1)-induced progesterone and estradiol production by large- and small-follicle GCs, but the inhibitory effect of BMP4 on estradiol production was much more pronounced in large-follicle GCs. In small-follicle GCs, BMP4 had no effect (P > 0.10) on IGF1-induced proliferation, but GREM inhibited (P < 0.05) cell proliferation and estradiol and progesterone production in IGF1 plus FSH-treated GCs. In large-follicle GCs, BMP4 (10 to 30 ng/mL) increased (P < 0.05) GC numbers and GREM (100 ng/mL) blocked this effect. In large-follicle GCs, CTGF inhibited (P < 0.05) FSH plus IGF1-induced progesterone and estradiol production, and CTGF blocked the stimulatory effect of BMP4 on GC proliferation. These results indicate that BMP4, GREM, and CTGF inhibit GC aromatase activity and progesterone production. Also, the stimulatory effect of BMP4 on GC proliferation and the inhibitory effects of BMP4 on GC steroidogenesis are more pronounced in large vs. small follicles.

Objective To investigate the presence of WNT antagonists Dickkopf-related protein 1 (DKK1), Frizzled-related protein (FRZB) and BMP antagonist Gremlin 1 (GREM1) in synovial fluid (SF) and serum, respectively, from end-stage knee osteoarthritis (OA) patients, and correlate their expression with other markers of OA. Design In a cross-sectional study, SF and serum were collected from OA patients ( n = 132). The concentrations of DKK1, FRZB and GREM1 in SF and serum were determined using immunoassays. Correlation measurements were performed between groups and previously assessed disease markers, such as synovium nitric oxide (NO), inerleukin-1β (IL1β), tumor necrosis factor–α (TNFα), and prostaglandin E2 (PGE2). Results The OA patients with the celecoxib treatment till surgery have higher median SF FRZB values compared with the control (no treatment); the celecoxib 3-days before surgery stopped treatment group has higher median serum FRZB values than the control and the naproxen treatment group. The combinational analysis of SF DKK1 and SF FRZB negatively correlated with macroscopic cartilage scores and histological synovium scores in OA patients. The expression of DKK1 and FRZB in SF showed the same expression trend as their expression in serum. Furthermore, the SF concentration of DKK1 was positively correlated with FRZB in both SF and serum. In contrast, it was negatively correlated with synovium NO and IL1β. SF FRZB was negatively correlated with synovium NO, IL1β, cartilage PGE2, and age. Conclusions Our findings suggest DKK1 and FRZB were negatively correlated with OA severity and multiple pro-inflammatory cytokines. Our data indicate that DKK1 and FRZB can be joint disease–specific biomarkers.

Bone morphogenetic protein (BMP) signaling is required for early forebrain development and cortical formation. How the endogenous modulators of BMP signaling regulate the structural and functional maturation of the developing brain remains unclear. Here, we show that expression of the BMP antagonist Grem1 marks committed layer V and VI glutamatergic neurons in the embryonic mouse brain. Lineage tracing of Grem1-expressing cells in the embryonic brain was examined by administration of tamoxifen to pregnant Grem1creERT; Rosa26LSLTdtomato mice at 13.5 days post coitum (dpc), followed by collection of embryos later in gestation. In addition, at 14.5 dpc, bulk mRNA-seq analysis of differentially expressed transcripts between FACS-sorted Grem1-positive and -negative cells was performed. We also generated Emx1-cre-mediated Grem1 conditional knockout mice (Emx1-Cre;Grem1flox/flox) in which the Grem1 gene was deleted specifically in the dorsal telencephalon. Grem1Emx1cKO animals had reduced cortical thickness, especially layers V and VI, and impaired motor balance and fear sensitivity compared with littermate controls. This study has revealed new roles for Grem1 in the structural and functional maturation of the developing cortex., Summary: The BMP antagonist Grem1 is expressed by committed deep-layer glutamatergic neurons in the embryonic mouse cortex. Grem1 conditional knockout mice display cortical and behavioral abnormalities.

ObjectiveSystemic Sclerosis is an autoimmune connective tissue disease which results in fibrosis of the skin and lungs. The disease is characterized by activation of myofibroblasts but what governs this is unknown. Gremlin-1 is a BMP antagonist that is developmentally regulated and we sought to investigate its role in Systemic Sclerosis.MethodsDermal fibroblasts were transfected with Grem1pcDNA3.1 expression vectors or empty vectors. Various markers of myofibroblasts were measured at the mRNA and protein levels. Scratch wound assays were also performed. Media Transfer experiments were performed to evaluate cytokine like effects. Various inhibitors of TGF-β signaling and MAPK signaling were used post-transfection. siRNA to Gremlin-1 in SSc dermal fibroblasts were performed to evaluate the role of Gremlin-1. Different cytokines were incubated with fibroblasts and Gremlin-1 measured. Bleomycin was used as model of fibrosis and immunohistochemistry performed.ResultsOverexpression of Gremlin-1 was achieved in primary dermal fibroblasts and lead to activation of quiescent cells to myofibroblasts indicated by collagen and α-Smooth muscle actin. Overexpression also led to functional effects. This was associated with increased TGF-β1 levels and SBE luciferase activity but not increased Thrombospondin-1 expression. Inhibition of Gremlin-1 overexpression cells with antibodies to TGF-β1 but not isotype controls led to reduced collagen and various TGF-β pathway chemical inhibitors also led to reduced collagen levels. In SSc cells siRNA mediated reduction of Gremlin-1 reduced collagen expression and CTGF gene and protein levels in these cells. IL-13 did not lead to elevated Gremlin-1 expression nor did IL-11. Gremlin-1 was elevated in an animal model of fibrosis compared to NaCl-treated mice.ConclusionGremlin-1 is a key regulator of myofibroblast transition leading to enhanced ECM deposition. Strategies that block Gremlin-1 maybe a possible therapeutic target in fibrotic diseases such as SSc.

Background: Available evidences have shown that bone morphogenetic proteins (BMPs), particularly BMP 2 and BMP 4, are involved in vascular calcification. Gremlin 1 is one of the important endogenous inhibitors of BMPs. This extracellular antagonist of bone morphogenic proteins has a very complex and cysteine-rich chain and establishes non-colonial transmissions to the members of the family with varying degrees of dependence and prevents them from binding to the receptor, thereby inhibit their function. The main objective of this study was clinical evaluation of the correlation between Gremlin-1 serum concentration and Coronary Artery Calcification. Methods: Eighty-four patients with coronary artery disease from cardiology ward of Razavi Hospital, Mashhad, Iran, who completed the inclusion criteria, entered in the study between November 2015 and March 2016. CT-Angiography was performed to define coronary artery calcium score and Gremlin-1 serum concentrations were measured by an ELISA kit. Results: Eighty-one patients, with mean age of 57.19±10.18 years were included to the study. The mean serum level of Gremlin-1 was 10.92±8.46 pg/mL. There was a reverse significant correlation between Gremlin-1 serum concentration and Coronary Artery Calcification of Right coronary artery(RCA) (P

Aim Branched-chain amino acids (BCAAs) have been reported owning curative effects in early diabetic nephropathy. However, the mechanisms of its action have not been elucidated. The aim of this study is to investigate the effect of possible mechanism(s) of BCAAs on cultured rat mesangial cells (RMCs). Methods RMCs were treated with high glucose (30 mmol/L) and BCAAs (10 mmol/L) respectively. Cell proliferation was detected using an MTT assay. Expression of transforming growth factor (TGF)-β1 and gremlin mRNA was detected by semiquantitative reverse-transcription (RT) PCR. TGF-β1 and fibronectin (FN) protein levels were measured using enzyme-linked immunosorbent assays (ELISAs). Gremlin, bone morphogenic protein (BMP)-7, and Smad2/3 proteins were detected by immunofluorescence. Smad1/5/8 and phosphorylated (p)-Smad1/5/8 were detected by Western blotting. Results The proliferation rate of the RMCs in the high glucose group alone was 1.45-times of cells in the CON group, and it was reduced by 32% upon co-treatment with BCAAs. The expression of TGF-β1, gremlin, p-Smd2/3 and FN mRNA or protein in the HG group was higher than that in the CON group. In the BCAAs group, the corresponding levels were lower than that in HG group. The expression of BMP-7 and p-Smad1/5/8 were significantly lower in the HG group than in the CON group. Moreover, the expression of BMP-7 and p-Smad1/5/8 were higher in the BCAAs group than in the HG group. Conclusion BCAAs showed an antidiabetic effect via reducing TGF-β1-Smad2/3 pathway and Gremlin expression and upregulating BMP-7-Smad1/5/8 pathway in rat mesangial cells, consequently lessening ECM deposition in renal tissue.

Gremlin renal overexpression has been reported in diabetic nephropathy, pauci-immune crescentic glomerulonephritis and chronic allograft nephropathy and has been implicated in the pathophysiology of the progression of renal damage. However, it is unknown whether urinary Gremlin can be associated with renal functional status, renal biopsy findings and outcome. To examine these associations we studied 20 patients with ANCA+ renal vasculitis and very high urinary Gremlin (354 ± 76 ug/gCr), 86 patients with other glomerular diseases and moderately elevated urinary Gremlin (83 ± 14 ug/gCr) and 11 healthy controls (urinary Gremlin 11.3 ± 2.4 ug/gCr). Urinary Gremlin was significantly correlated with renal expression of Gremlin (r = 0.64, p = 0.013) observed in cellular glomerular crescents, tubular epithelial cells and interstitial inflammatory cells. Moreover, urinary Gremlin levels were correlated with the number of glomerular crescents (r = 0.53; p < 0.001), renal CD68 positive cells (r = 0.71; p < 0.005), tubulointerstitial fibrosis (r = 0.50; p < 0.05), and serum creatinine levels (r = 0.60; p < 0.001). Interestingly, Gremlin expression was colocalized with CD68, CD163 (monocyte/macrophage markers) and CCL18 positive cells. ROC curve analysis showed that the cutoff value of urinary Gremlin in glomerular diseases as 43 ug/gCr with 72% of sensitivity and 100% of specificity [AUC: 0.96 (CI 95% 0.92–0.99] (p < 0.001). For ANCA+ renal vasculitis the value of urinary Gremlin of 241 ug/gCr had 55% of sensitivity and 100% of specificity [AUC: 0.81 (CI 95% 0.68–0.94) (p < 0.001]. Based on these results we propose that urinary Gremlin represents a non-invasive biomarker in ANCA+ renal vasculitis, and suggest a role of Gremlin in the formation of crescents., This work was supported by the Fondecyt 1160465 (Chile) Proyecto PAI 82140017, Fondecyt de Iniciación 111-40242, and Division of Nephrology, Universidad Austral Valdivia, Chile; Instituto de Salud Carlos III and Fondos FEDER European Union (PI014/0041, PI14/00386, PI17/01495, PI17/00119), Red de Investigación Renal (REDinREN; RD16/009), Comunidad de Madrid (NOVELRENCM (B2017/BMD3751)

Introduction: Systemic sclerosis is an autoimmune connective tissue disease in which there is activation of the immune system, vascular disease and fibrosis. Activation of quiescent fibroblasts to myofibroblasts is key to disease pathogenesis. Gremlin-1 is a bone morphogenetic protein antagonist which is important in development and we recently reported in skin fibrosis. The aim of this study was to determine the serum circulating levels of Gremlin-1 in early diffuse systemic sclerosis. Methods: Twenty-one early diffuse systemic sclerosis patients (less than 2 years from first non-Raynaud’s symptom) were included and age and sex-matched healthy controls. Serum was isolated from blood and measured with a specific enzyme-linked immunoassay for Gremlin-1. Clinical variables were also measured. Results: Significantly elevated Gremlin-1 was found in sera of early diffuse systemic sclerosis patients ( p < 0.001). In patients with interstitial lung disease, this compared to systemic sclerosis without evidence of interstitial lung disease, Gremlin-1 was significantly elevated ( p < 0.0007). A correlation was found between circulating Gremlin-1 and modified Rodnan Skin Score, albeit weak. Discussion: In early diffuse systemic sclerosis patients, elevated Gremlin-1 is found in serum. This is particularly prominent in systemic sclerosis–associated interstitial lung disease. This suggests that Gremlin-1 may be a biomarker for systemic sclerosis interstitial lung disease.

Background He-Chan Pian (HCP), a traditional Chinese medicinal formula, shows promising efficacy for the treatment of lung cancer. Purpose Gremlin (GREM1) plays an important role in gastrointestinal tumor metastasis; however, little is known about its role in lung cancer. We determined the mechanism underlying the protective effect of HCP against metastasis in a mouse model of non-small cell lung cancer (NSCLC) and demonstrated the role of GREM1. Methods Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) was used to analyze the herbal components and metabolites from the serum of HCP-treated mice. The tumor, liver, and kidney were examined histologically, and the antitumor effects and toxicity of HCP were evaluated. Levels of epithelial-mesenchymal transition (EMT)-associated transcription factors were measured using western blotting in tumors from five groups (i.e., model, HCP [L], HCP [M], HCP [H], and positive control [cisplatin, DDP]). Differentially expressed proteins and genes were identified using protein chip and sequencing analyzes, respectively. Short hairpin RNAs and overexpression plasmids were introduced into cells to evaluate the effects of GREM1. To evaluate proliferation, migration, and invasion, the expression levels of proteins involved in the Rap1 pathway and EMT were measured in vitro. Xenograft tumors with overexpression-GREM1 (OE-GREM1) in A549 cells were examined for cell proliferation. A dual-luciferase assay was performed to verify the direct interaction of GREM1 with miR-205-5p in lung cancer. Results Thirty-six ingredients and bioactive constituents detected in the serum of HCP-treated mice were identified as the key compounds involved in the inhibition of tumor growth. Animal experiments revealed that HCP significantly decreased tumor volumes and had no adverse effects on the liver or kidney or side effects. GREM1 upregulation was closely related to tumor metastasis and was regulated by miR-205-5p, as confirmed using a dual-luciferase reporter assay. OE-GREM1 promoted A549 cell migration and invasion, promoted EMT, and increased the expression of Rap1 pathway intermediaries, whereas shGREM1 had the opposite effects. Furthermore, the effects of OE-GREM1 on proliferation in the A549 xenograft mouse model were attenuated, although HCP has an inhibitory effect on tumors. Conclusion Our results suggest that HCP contributes to the inhibition of NSCLC metastasis via the Gremlin/Rap1 signaling pathway regulated by miR-205-5p.

Intestinal fibrosis is a consequence of continuous inflammatory responses that negatively affect the quality of life of patients. By screening altered proteomic profiles of mouse fibrotic colon tissues, we identified that GREM1 was dramatically upregulated in comparison to that in normal tissues. Functional experiments revealed that GREM1 promoted the proliferation and activation of intestinal fibroblast cells by enhancing fatty acid oxidation. Blocking GREM1 prevented the progression of intestinal fibrosis in vivo. Mechanistic research revealed that GREM1 acted as a ligand for VEGFR2 and triggered downstream MAPK signaling. This facilitated the expression of FAO-related genes, consequently enhancing fatty acid oxidation. Taken together, our data indicated that targeting GREM1 could represent a promising therapeutic approach for the treatment of intestinal fibrosis.

Introduction HER2 over-expression denotes poor prognosis in breast cancers.Bone morphogenetic protein(BMP) signalling is known to interact with EGF signalling, co-regulating breast cancer progression.BMP antagonist Gremlin-1 may influence breast cancer disease progression, but this remains unexplored in HER2 positive breast cancers. Method GREM1 and HER2 expression, and clinical outcomes were examined in clinical cohorts.GREM1 overexpression or pEF control plasmid were transduced into BT474 HER2+breast cancer cells. In vitro function tests using BT474 pEF and BT474GREM1cells include 2D/3D growth, migration, and expression of epithelial to mesenchymal transition(EMT)markers. Signalling cascades were examined in BT474 treated with RhGremlin-1. In vivo, BALB/c nude mice underwent either mammary injection or intra-cardiac injection of BT474pEF or BT474GREM1 cells and disease burden assessed. Result GREM1 expression correlates with HER2 in breast tumours(p=0.03) and is higher in metastatic HER2 positive cancers (p = 0.04). HER2 positive patients with high GREM1 have poor survival(p = 0.0002). BT474GREM1cells have up-regulated markers of EMT compared to control. BT474 RhGremlin-1 treated cells have active AKT pathway signalling, independent of BMP signalling. In vitro, BT474GREM1cells significantly proliferate and migrate compared to control(p Conclusion Gremlin-1 is correlated with poor outcomes in HER2 patients and promotes breast cancer cellular growth, migration and metastasis.Gremlin-1 is a novel area of research with potential as a prognostic biomarker and therapeutic target for personalised, effective, breast cancer outcomes. Take-home message BMP antagonists are gaining interest for their potential in breast cancer prognosis and therapeutics.This novel area of research shows BMP antagonist Gremlin-1 is of importance in HER2 positive breast cancers. DRAGONS DEN

Objective Gremlin 1 (GREM1) is a secreted BMP2/4 inhibitor which regulates commitment and differentiation of human adipose precursor cells and prevents the browning effect of BMP4. GREM1 is an insulin antagonist and serum levels are high in type 2 diabetes (T2D). We here examined in vivo effects of AAV8 (Adeno-Associated Viral vectors of serotype eight) GREM 1 targeting the liver in mature mice to increase its systemic secretion and also, in a separate study, injected recombinant GREM 1 intraperitoneally. The objective was to characterize systemic effects of GREM 1 on insulin sensitivity, glucose tolerance, body weight, adipose cell browning and other local tissue effects. Methods Adult mice were injected with AAV8 vectors expressing GREM1 in the liver or receiving regular intra-peritoneal injections of recombinant GREM1 protein. The mice were fed with a low fat or high fat diet (HFD) and followed over time. Results Liver-targeted AAV8-GREM1 did not alter body weight, whole-body glucose and insulin tolerance, or adipose tissue gene expression. Although GREM1 protein accumulated in liver cells, GREM1 serum levels were not increased suggesting that it may not have been normally processed for secretion. Hepatic lipid accumulation, inflammation and fibrosis were also not changed. Repeated intraperitoneal rec-GREM1 injections for 5 weeks were also without effects on body weight and insulin sensitivity. UCP1 was slightly but significantly reduced in both white and brown adipose tissue but this was not of sufficient magnitude to alter body weight. We validated that recombinant GREM1 inhibited BMP4-induced pSMAD1/5/9 in murine cells in vitro, but saw no direct inhibitory effect on insulin signalling and pAkt (ser 473 and thr 308) activation. Conclusion GREM1 accumulates intracellularly when overexpressed in the liver cells of mature mice and is apparently not normally processed/secreted. However, also repeated intraperitoneal injections were without effects on body weight and insulin sensitivity and adipose tissue UCP1 levels were only marginally reduced. These results suggest that mature mice do not readily respond to GREMLIN 1 but treatment of murine cells with GREMLIN 1 protein in vitro validated its inhibitory effect on BMP4 signalling while insulin signalling was not altered.

Members of the differential screening-selected gene aberrative in neuroblastoma (DAN) protein family are developmentally conserved extracellular binding proteins that antagonize bone morphogenetic protein (BMP) signaling. This protein family includes the Gremlin proteins, GREM1 and GREM2, which are known to have key functions during embryogenesis and adult physiology. While BMPs play essential roles in adult female reproductive physiology, the role of the DAN family in ovarian function is less understood. We generated mice null forGrem2to study its role in female fertility in addition to screening patients with primary ovarian insufficiency for variants in GREM2.Grem2-/-mice are viable and femaleGrem2-/-mice have diminished fecundity and irregular estrous cycles. This is accompanied by reduced serum levels of anti-Müllerian hormone, a marker of the ovarian reserve, in adult mice. Alterations in ovarian expression of inhibin and activin subunit genes, which are required for regulation of the hypothalamic-pituitary-ovarian (HPO) axis, were identified. WhileGrem2mRNA transcript was not detected in the pituitary,Grem2was expressed in the hypothalami of wild type female mice. Additionally, screening 106 women with primary ovarian insufficiency identified one individual with a heterozygous variant in GREM2 that lies within the predicted BMP-GREM2 interface. In total, these data suggest thatGrem2is necessary for female fecundity by playing a novel role in regulating the HPO axis and possibly contributing to female reproductive disease.

Objective. To investigate the function of NAT10 in mesenchymal stem cell (MSC) osteogenic differentiation and study the mechanism by which NAT10 affects MSC osteogenesis by mediating Gremlin 1 N4-acetylcytidine (ac4C) modification. Methods. Osteogenic differentiation of MSCs was induced, and the osteogenic ability was evaluated with alizarin red S (ARS) and alkaline phosphatase (ALP) assays. The NAT10 expression level during MSC osteogenesis was measured by western blot (WB). MSCs were transfected with lentiviruses to inhibit (Sh-NAT10) or overexpress NAT10 (Over-NAT10), and the osteogenic differentiation ability was assessed by ARS, ALP, and osteogenic gene marker assays. β-Catenin, Akt, and Smad signaling pathway component activation levels were assessed, and the expression levels of key Smad signaling pathway molecules were determined by PCR and WB. The Gremlin 1 mRNA ac4C levels were analyzed using RIP-PCR, and the Gremlin 1 mRNA degradation rate was determined. Sh-Gremlin 1 was transfected to further investigate the role of NAT10 and Gremlin 1 in MSC osteogenesis. Results. During MSC osteogenesis, NAT10 expression, ARS staining, and the ALP level gradually increased. Decreasing NAT10 expression inhibited, and increasing NAT10 expression promoted MSC osteogenic differentiation. NAT10 affected the BMP/Smad rather than the Akt and β-Catenin signaling pathway activation by regulating Gremlin 1 expression. The Gremlin 1 mRNA ac4C level was positively regulated by NAT10, which accelerated Gremlin 1 degradation. Sh-Gremlin 1 abolished the promotive effect of NAT10 on MSC osteogenic differentiation. Conclusion. NAT10 positively regulated MSC osteogenic differentiation through accelerating the Gremlin 1 mRNA degradation by increasing its ac4C level. These results may provide new mechanistic insight into MSC osteogenesis and bone metabolism in vivo.

Gremlin-1 (GREM1), one of the antagonists of bone morphogenetic proteins (BMPs), has recently been reported to be overexpressed in a variety of cancers including breast cancer. GREM1 is involved in tumor promotion, but little is known about its role in the glycolysis of cancer cells. In this study, we investigated the role of GREM1 in glycolysis of breast cancer cells and its underlying molecular mechanisms. We first observed that glucose uptake and lactate production were increased in GREM1-overexpressing breast cancer cells. GREM1 increased the expression of hexokinase-2 (HK2), which catalyzes the phosphorylation of glucose, the first step in glycolysis. In addition, GREM1 activated STAT3 transcription factor through the ROS-Akt signaling pathway. The ROS-Akt-STAT3 axis activated by GREM1 was involved in promoting glucose uptake by increasing the expression of HK2 in breast cancer cells. Therefore, our study suggested a new mechanism by which GREM1 is involved in breast cancer promotion by increasing glycolysis in breast cancer cells.

Background Heterozygous mutation in BMP (bone morphogenetic protein) receptor 2 is rare, but BMP cascade suppression is common in congenital heart disease–associated pulmonary arterial hypertension (CHD‐PAH); however, the underling mechanism of BMP cascade suppression independent of BMP receptor 2 mutation is unknown. Methods and Results Pulmonary hypertensive status observed in CHD‐PAH was surgically reproduced in rats. Gremlin‐1 expression was increased, but BMP cascade was suppressed, in lungs from CHD‐PAH patients and shunted rats, whereas shunt correction retarded these trends in rats. Immunostaining demonstrated increased gremlin‐1 was mainly in the endothelium and media of remodeled pulmonary arteries. However, mechanical stretch time‐ and amplitude‐dependently stimulated gremlin‐1 secretion and suppressed BMP cascade in distal pulmonary arterial smooth muscle cells from healthy rats. Under static condition, gremlin‐1 significantly promoted the proliferation and inhibited the apoptosis of distal pulmonary arterial smooth muscle cells from healthy rats via BMP cascade. Furthermore, plasma gremlin‐1 closely correlated with hemodynamic parameters in CHD‐PAH patients and shunted rats. Conclusions Serving as an endogenous antagonist of BMP cascade, the increase of gremlin‐1 in CHD‐PAH may present a reasonable mechanism explanation for BMP cascade suppression independent of BMP receptor 2 mutation.

Hydra, a Cnidarian believed to have been evolved about 60 million years ago, has been a favorite model for developmental biologists since Abraham Trembley introduced it in 1744. However, the modern renaissance in research on hydra was initiated by Alfred Gierer when he established a hydra laboratory at the Max Plank Institute in Göttingen in the late 1960s. Several signaling mechanisms that regulate development and pattern formation in vertebrates, including humans, have been found in hydra. These include Wnt, BMP, VEGF, FGF, Notch, and RTK signaling pathways. We have been using hydra to understand the evolution of cell signaling for the past several years. In this article, I will summarize the work on cell signaling pathways in hydra with emphasis on our own work. We have identified and characterized, for the first time, the hydra homologs of the BMP inhibitors Noggin and Gremlin, the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and several receptor tyrosine kinases (RTKs). Our work, along with that of others, clearly demonstrates that these pathways arose early in evolution to carry out functions that were often quite different from their functions in more complex animals. Apart from providing insights into morphogenesis and pattern formation in adult, budding and regenerating hydra, these findings bring out the utility of hydra as a model system to study evolutionarily ancient, in contrast to recently acquired, functions of various biological molecules.

Aims Circ_0020397 has been found to be down-regulated in intracranial aneurysm (IA), and deregulation of circ_0020397 involved in the regulation of vascular smooth muscle cells (VSMCs) proliferation. However, the mechanism by which circ_0020397 implicates in VSMC dysfunction in IA remains vague. Materials and methods The expression of circ_0020397, miR-502-5p and Gremlin 1 (GREM1) was detected using quantitative real-time polymerase chain reaction. Cell viability was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Protein levels of proliferating cell nuclear antigen (PCNA) and GREM1 were measured using western blot. The interaction between miR-502-5p and circ_0020397 or GREM1 was confirmed by dual-luciferase reporter, RNA pull-down and RNA immunoprecipitation assay. Key findings Circ_0020397 or GREM1 expression was decreased in VSMCs isolated from IA patients, and overexpression of circ_0020397 or GREM1 promoted VSMC viability and elevated PCNA expression level, while inhibition of them showed opposite effects. MiR-502-5p was confirmed to directly bind to circ_0020397 or GREM1, and miR-502-5p reversed the effects of circ_0020397 on VSMC viability and PCNA level. Besides, miR-502-5p overexpression suppressed VSMC viability and reduced PCNA level, while these effects were attenuated by GREM1 up-regulation. Importantly, circ_0020397 could regulate GREM1 expression via miR-502-5p in VSMCs. Significance Circ_0020397 played an important role in phenotypic modulation in IA by promoting VSMC viability via miR-502-5p/GREM1 axis, suggesting a novel insight into IA pathogenesis and new targets for IA molecular therapy.

The bone morphogenetic proteins (BMPs), a subgroup of the transforming growth factor-β (TGF-β) superfamily, are involved in multiple biological processes such as embryonic development and maintenance of adult tissue homeostasis. The importance of a functional BMP pathway is underlined by various diseases, including cancer, which can arise as a consequence of dysregulated BMP signaling. Mutations in crucial elements of this signaling pathway, such as receptors, have been reported to disrupt BMP signaling. Next to that, aberrant expression of BMP antagonists could also contribute to abrogated signaling. In this review we set out to highlight how BMP antagonists affect not only the cancer cells, but also the other cells present in the microenvironment to influence cancer progression.

Computational modeling can be used to investigate complex signaling networks in biology. However, most modeling tools are not suitable for molecular cell biologists with little background in mathematics. We have built a visual-based modeling tool for the investigation of dynamic networks. Here, we describe the development of computational models of cartilage development and osteoarthritis, in which a panel of relevant signaling pathways are integrated. In silico experiments give insight in the role of each of the pathway components and reveal which perturbations may deregulate the basal healthy state of cells and tissues. We used a previously developed computational modeling tool Analysis of Networks with Interactive Modeling (ANIMO) to generate an activity network integrating 7 signal transduction pathways resulting in a network containing over 50 nodes and 200 interactions. We performed in silico experiments to characterize molecular mechanisms of cell fate decisions. The model was used to mimic biological scenarios during cell differentiation using RNA-sequencing data of a variety of stem cell sources as input. In a case-study, we wet-lab-tested the model-derived hypothesis that expression of DKK1 (Dickkopf-1) and FRZB (Frizzled related protein, WNT antagonists) and GREM1 (gremlin 1, BMP antagonist) prevents IL1β (Interleukin 1 beta)-induced MMP (matrix metalloproteinase) expression, thereby preventing cartilage degeneration, at least in the short term. We found that a combination of DKK1, FRZB and GREM1 may play a role in modulating the effects of IL1β induced inflammation in human primary chondrocytes. ispartof: CELLULAR SIGNALLING vol:68 ispartof: location:England status: published

Background Mechanisms regulating BMP and Wnt pathways and their interactions are not well studied in Hydra. Results We report identification of BMP inhibitor gremlin, comparison of its expression with that of noggin and possible antagonism between Wnt and BMP signaling in Hydra. Gremlin is expressed in body column with high levels in budding region and in early buds. Noggin, on the other hand, is expressed in the hypostome, base of tentacles, lower body column, and basal disc. During budding, noggin is expressed at the sites of tentacle emergence. This was confirmed in ectopic tentacles in polyps treated with alsterpaullone (ALP), a GSK-3β inhibitor that leads to upregulation of Wnt pathway. RT-PCR data show that upregulation of Wnt is accompanied by downregulation of bmp 5-8b though noggin and gremlin remain unaltered till 24 hours. Conclusions Different expression patterns of gremlin and noggin suggest their roles in budding and patterning of tentacles, respectively. Further, bmp 5-8b inhibition by activated Wnt signaling does not directly involve noggin and gremlin in Hydra. Our data suggest that Wnt/BMP antagonism may have evolved early for defining the oral-aboral axis, while the involvement of BMP antagonists during axial patterning is a recent evolutionary acquisition within the Bilateria lineage.

Background: diabetic nephropathy (DN) is the most common cause of end-stage of renal disease. It is beneficial for us to find effective way to treat the disease. Gremlin is deemed as a key factor in the development of diabetic nephropathy at present. We hypothesized that the pathological changes might be prevented by eliminating Gremlin function in high glucose-induced renal fibrosis. Methods: lentiviral vector targeting Gremlin was applied to inhibit Gremlin expression at a high glucose concentration which simulated diabetic nephropathy in rat mesangial cells. Results: the shRNA vector, designated shGremlin, significantly inhibited Gremlin expression in rat mesangial cells cultured under high glucose conditions. Increase in BMP-7 as well as its downstream genes or proteins including phospholated Smad 1/5/8, type IV collagen and fibronectin was observed. Conclusions: our work provides a valuable method to prevent glomerular/renal fibrosis with RNA interference, and also enable development of new therapies that target Gremlin.

Gremlin is a member of the TGF-β superfamily that can act as a BMP antagonist, and recently, has been described as a ligand of the vascular endothelial growth factor receptor 2 (VEGFR2). Gremlin shares properties with the Notch signaling pathway. Both participate in embryonic development and are reactivated in pathological conditions. Gremlin is emerging as a potential therapeutic target and biomarker of renal diseases. Here we review the role of the Gremlin-VEGFR2 axis in renal damage and downstream signaling mechanisms, such as Notch pathway.

Diabetic retinopathy (DR) is one of the most common causes of blindness globally. Proliferative DR (PDR), an advanced stage of DR, is characterized by the formation of fibrotic membranes at the vitreoretinal interface. The proliferation, migration, and secretion of extracellular matrix molecules in retinal pigment epithelial (RPE) cells contribute to the formation of fibrotic membranes in PDR. Gremlin has been reported to be upregulated in response to elevated glucose levels in the retina of diabetic rat and bovine pericytes. However, the role of gremlin in PDR remains unclear. In the present study, the vitreous concentrations of gremlin were significantly higher in the PDR (67.79±33.96) group than in the control (45.31±12.31) group, and high glucose levels induced the expression of gremlin in RPE cells. The elevated expression of extracellular matrix molecules, such as fibronectin and collagen IV, was significantly reduced by gremlin siRNA in human RPE cells under high-glucose conditions. Thus, gremlin may play a vital role in the development of PDR.

The absence of optimal treatments for Diabetic Nephropathy (DN) highlights the importance of the search for novel therapeutic targets. The vascular endothelial growth factor receptor 2 (VEGFR2) pathway is activated in experimental and human DN, but the e ects of its blockade in experimental models of DN is still controversial. Here, we test the e ects of a therapeutic anti-VEGFR2 treatment, using a VEGFR2 kinase inhibitor, on the progression of renal damage in the BTBR ob/ob (leptin deficiency mutation) mice. This experimental diabetic model develops histological characteristics mimicking the key features of advanced human DN. A VEGFR2 pathway-activation blockade using the VEGFR2 kinase inhibitor SU5416, starting after kidney disease development, improves renal function, glomerular damage (mesangial matrix expansion and basement membrane thickening), tubulointerstitial inflammation and tubular atrophy, compared to untreated diabetic mice. The downstream mechanisms involved in these beneficial e ects of VEGFR2 blockade include gene expression restoration of podocyte markers and downregulation of renal injury biomarkers and pro-inflammatory mediators. Several ligands can activate VEGFR2, including the canonical ligands VEGFs and GREMLIN. Activation of a GREMLIN/VEGFR2 pathway, but not other ligands, is correlated with renal damage progression in BTBR ob/ob diabetic mice. RNA sequencing analysis of GREMLIN-regulated genes confirm the modulation of proinflammatory genes and related-molecular pathways. Overall, these data show that a GREMLIN/VEGFR2 pathway activation is involved in diabetic kidney disease and could potentially be a novel therapeutic target in this clinical condition, This research was funded by Fondecyt 1160465 to S.M., e Instituto de Salud Carlos III (ISCIII) and Fondos FEDER European Union (Grants PI17/00119 and Red de Investigación Renal REDINREN: RD16/0009 to M.R.-O; and PI17/01495 to J.E.). Comunidad de Madrid (Grant “NOVELREN” B2017/BMD-3751 to M.R.-O).

Bone morphogenetic protein (BMP) signalling is key to many developmental processes, including early regionalisation of the ectoderm. The neural crest is induced here by a combination of BMP and Wnt signals from nearby tissues with many secreted factors contributing to its initial specification at the neural plate border. Gremlin 1 (Grem1) is a secreted BMP antagonist expressed in the neural crest in Xenopus laevis but its function here is unknown. As well as binding BMPs, Grem1 has been shown to interact with heparan sulfate proteoglycans (HSPGs), a family of cell surface macromolecules that regulate a diverse array of signalling molecules by affecting their availability and mode of action. This study describes the impact of HSPGs on the function of Grem1 in neural crest induction. It shows for the first time that Grem1 is required for neural crest development in a two-step process comprising an early HSPG-independent, followed by a late HSPG-dependent phase., Highlights • Gremlin 1 is required for neural crest and neural plate border specification. • HSPGs regulate Gremlin 1 activity in vivo. • Neural crest induction has HSPG independent and dependent phases.

Background/Aims: The transplantation of cardiac progenitor cells (CPCs) improves neovascularization and left ventricular function after myocardial infarction (MI). The bone morphogenetic protein antagonist Gremlin 2 (Grem2) is required for early cardiac development and cardiomyocyte differentiation. The present study examined the role of Grem2 in CPC differentiation and cardiac repair. Methods: To determine the role of Grem 2 during CPC differentiation, c-Kit+ CPCs were cultured in differentiation medium for different times, and Grem2, Notch1 and Jagged1 expression was determined by RT-PCR and western blotting. Short hairpin RNA was used to silence Grem2 expression, and the expression of cardiomyocyte surface markers was assessed by RT-PCR and immunofluorescence staining. In vivo experiments were performed in a mouse model of left anterior descending coronary artery ligation-induced MI. Results: CPC differentiation upregulated Grem2 expression and activated the Notch1 pathway. Grem2 knockdown inhibited cardiomyocyte differentiation, and this effect was similar to that of Notch1 pathway inhibition in vitro. Jagged1 overexpression rescued the effects of Grem2 silencing. In vivo, Grem2 silencing abolished the protective effects of CPC injection on cardiac fibrosis and function. Conclusions: Grem2 regulates CPC cardiac differentiation by modulating Notch1 signaling. Grem2 enhances the protective effect of CPCs on heart function in a mouse model of MI, suggesting its potential as the rapeutic protein for cardiac repair.

The antagonist-specific regulation in tissue engineering constitutes important attempts to achieve an improved and rapid bone regeneration by controlling the natural biological response of the natural body growth factors. L51P is molecularly engineered bone morphogentic protein-2 (BMP-2) variant with a substitution of the 51st leucine with a proline residue. L51P is deficient in BMP receptor binding, but maintains its structure and affinity for inhibitory proteins such as noggin, chordin, and gremlin. These modifications convert the BMP-2 variant L51P into a receptor-inactive inhibitor of BMP antagonists. This current approach may prevent the uncontrolled bone overgrowth using high concentration of BMPs and thus regulates the possible growth factor's high-dose side effects. Exploring of L51P biological functions is required to broad our understanding of BMP mutant biological functions and their potential clinical applications. The progress of L51P researches would hopefully lead to the development of multiple applications for using the L51P in bone and fracture healing disorders.

Slit guidance ligand 2 (SLIT2) is a large, secreted protein that binds roundabout (ROBO) receptors on multiple cell types, including neurons and kidney podocytes. SLIT2-ROBO–mediated signaling regulates neuronal migration and ureteric bud (UB) outgrowth during kidney development as well as glomerular filtration in adult kidneys. Additionally, SLIT2 binds Gremlin, an antagonist of bone morphogenetic proteins (BMPs), and BMP–Gremlin signaling also regulates UB formation. However, direct cross-talk between the ROBO2–SLIT2 and BMP–Gremlin signaling pathways has not been established. Here, we report the discovery of negative feedback between the SLIT2 and BMP–Gremlin signaling pathways. We found that the SLIT2–Gremlin interaction inhibited both SLIT2–ROBO2 signaling in neurons and Gremlin antagonism of BMP activity in myoblasts and fibroblasts. Furthermore, BMP2 down-regulated SLIT2 expression and promoter activity through canonical BMP signaling. Gremlin treatment, BMP receptor inhibition, and SMAD family member 4 (SMAD4) knockdown rescued BMP-mediated repression of SLIT2. BMP2 treatment of nephron progenitor cells derived from human embryonic stem cells decreased SLIT2 expression, further suggesting an interaction between the BMP2–Gremlin and SLIT2 pathways in human kidney cells. In conclusion, our study has revealed direct negative cross-talk between two pathways, previously thought to be unassociated, that may regulate both kidney development and adult tissue maintenance.

Background/Aims: Bone nonunion remains a challenge for orthopaedists. The technological advancements that have been made in precisely silencing target genes have provided promising methods to address this challenge. Methods: We detected the expression levels of the bone morphogenetic protein (BMP) inhibitors Chordin, Gremlin and Noggin using realtime PCR in bone mesenchymal stem cells (BMSCs) isolated from patients with normal fracture healing and those with bone nonunion. Moreover, we detected the expression of Chordin, Gremlin and Noggin during the osteogenic differentiation of human BMSCs (hBMSCs) using real-time PCR and Western blot. We delivered Chordin siRNA to hBMSCs using a previously reported cationic polymer, polyspermine imidazole-4,5-imine (PSI), as a pH-responsive and non-cytotoxic transfection agent. The apoptosis and cellular uptake efficiency were analysed by flow cytometry. Results: We identified Chordin as the most appropriate potential therapeutic target gene for enhancing the osteogenic differentiation of hBMSCs. Chordin knockdown rescued the osteogenic capacity of hBMSCs isolated from patients with bone nonunion. Highly efficient knockdown of Chordin was achieved in hBMSCs using PSI. Chordin knockdown promoted hBMSC osteogenesis and bone regeneration in vitro and in vivo. Conclusions: Our results suggest that Chordin is a potential target for improving osteogenesis and bone nonunion therapy and that responsive and non-toxic cationic polyimines such as PSI are therapeutically feasible carriers for the packaging and delivery of Chordin siRNA to hBMSCs.

A paper in Nature Immunology describes a new subset of fibroblastic reticular cells, defined by expression of Gremlin 1, in lymphoid tissues that maintain homeostasis of conventional dendritic cells and ensure proper T cell immunity.

// Miao Yin 1, 2 , Mira Tissari 1, 2 , Jenni Tamminen 1, 2 , Irene Ylivinkka 1 , Mikko Ronty 3 , Pernilla von Nandelstadh 4 , Kaisa Lehti 4, 5, 6 , Marko Hyytiainen 4 , Marjukka Myllarniemi 7 and Katri Koli 1, 2 1 Research Programs Unit, Translational Cancer Biology, University of Helsinki, Helsinki, Finland 2 Transplantation Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland 3 Department of Pathology, University of Helsinki and Fimlab Laboratories, Pathology, Tampere, Finland 4 Research Programs Unit, Genome Scale Biology, University of Helsinki, Helsinki, Finland 5 K. Albin Johansson Foundation, Finnish Cancer Institute, Helsinki, Finland 6 Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Solna, Sweden 7 Department of Pulmonary Medicine, University of Helsinki and Helsinki University Hospital, Heart and Lung Center, Helsinki, Finland Correspondence to: Katri Koli, email: katri.koli@helsinki.fi Keywords: gremlin, mesothelioma, invasion Received: June 07, 2017 Accepted: September 21, 2017 Published: October 06, 2017 ABSTRACT Malignant mesothelioma originates from mesothelial cells and is a cancer type that aggressively invades into the surrounding tissue, has poor prognosis and no effective treatment. Gremlin-1 is a cysteine knot protein that functions by inhibiting BMP-pathway activity during development. BMP-independent functions have also been described for gremlin-1. We have previously shown high gremlin-1 expression in mesothelioma tumor tissue. Here, we investigated the functions of gremlin-1 in mesothelioma cell migration and invasive growth. Gremlin-1 promoted mesothelioma cell sprouting and invasion into three dimensional collagen and Matrigel matrices. The expression level of gremlin-1 was linked to changes in the expression of SNAI2, integrins, matrix metalloproteinases (MMP) and TGF-β family signaling - all previously associated with a mesenchymal invasive phenotype. Small molecule inhibitors of MMPs completely blocked mesothelioma cell invasive growth. In addition, inhibitors of TGF-β receptors significantly reduced invasive growth. This was associated with reduced expression of MMP2 but not SNAI2, indicating that gremlin-1 has both TGF-β pathway dependent and independent mechanisms of action. Results of in vivo mesothelioma xenograft experiments indicated that gremlin-1 overexpressing tumors were more vascular and had a tendency to send metastases. This suggests that by inducing a mesenchymal invasive cell phenotype together with enhanced tumor vascularization, gremlin-1 drives mesothelioma invasion and metastasis. These data identify gremlin-1 as a potential therapeutic target in mesothelioma.

Heart development depends on coordinated proliferation and differentiation of cardiac progenitor cells (CPCs), but how the two processes are synchronized is not well understood. Here, we show that the secreted Bone Morphogenetic Protein (BMP) antagonist GREMLIN 2 (GREM2) is induced in CPCs shortly after cardiac mesoderm specification during differentiation of human pluripotent stem cells. GREM2 expression follows cardiac lineage differentiation independently of the differentiation method used, or the origin of the pluripotent stem cells, suggesting that GREM2 is linked to cardiogenesis. Addition of GREM2 protein strongly increases cardiomyocyte output compared to established procardiogenic differentiation methods. Our data show that inhibition of canonical BMP signaling by GREM2 is necessary to promote proliferation of CPCs. However, canonical BMP signaling inhibition alone is not sufficient to induce cardiac differentiation, which depends on subsequent JNK pathway activation specifically by GREM2. These findings may have broader implications in the design of approaches to orchestrate growth and differentiation of pluripotent stem cell-derived lineages that depend on precise regulation of BMP signaling.

Gremlin-1, a bone morphogenetic protein (BMP) antagonist, has essential roles in kidney and limb bone development, and is important in chronic diseases including tissue fibrosis. It also functions as an activating ligand of the vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2), and binds strongly to the sulfated polysaccharide, heparin. Here we investigated the extent to which gremlin binds to the related polysaccharide heparan sulfate (HS), which unlike heparin is widely distributed spread within tissues. We determined that both highly sulfated HS and kidney HS are able to partially compete for the binding of heparin to gremlin, whereas low sulfated HS is a poor competitor. In further investigations of the interaction between gremlin and HS, we found that wild-type gremlin is able to bind broadly across the various regions of kidney in an HS-dependent manner, with particularly intense binding to tubular structures in the renal cortex. In a model of chronic kidney disease, fibrotic changes in the kidney result in a loss of gremlin binding sites. Gremlin mutants with reduced affinity for heparin showed negligible binding under the same conditions. These mutants nonetheless remain functional as BMP antagonists on C2C12 myoblastic cells transfected with a Smad 1 reporter gene construct. Overall our findings indicate that on secretion, gremlin will bind to HS structures on the cell surface and in the extracellular matrix, thus providing for a localised reservoir which can modulate BMP activity in a temporospatially restricted manner. Although binding of heparin/HS to gremlin has been shown elsewhere to be necessary for gremlin activation of VEGFR2, this does not appear to be essential for BMP antagonism by gremlin. Thus these sulfated polysaccharides differentially regulate the activities of gremlin.

Background Idiopathic pulmonary fibrosis (IPF) is a chronic and ultimately fatal disease characterized by a progressive decline in lung function. Fibrotic diseases, such as IPF, are characterized by uncontrolled activation of fibroblasts. Since the microenvironment is known to affect cell behavior, activated fibroblasts can in turn activate healthy neighboring cells. Thus, we investigated IPF paracrine signaling in human lung fibroblasts (HLFs) derived from patients with IPF. Methods Primary human fibroblast cultures from IPF (IPF-HLF) and control donor (N-HLF) lung tissues were established and their supernatants were collected. These supernatants were then added to N-HLFs for further culture. Protein and RNA were extracted from IPF/ N-HLFs at baseline. Interleukin-6 (IL-6) and TGF-β-related signaling factors (e.g. STAT3, Smad3) were evaluated by western blot and qPCR. IL-6 levels were measured by ELISA. IL-6 signaling was blocked by Tocilizumab (TCZ) (10 ng/ml). Results IPF-HLFs were found to significantly overexpress IL-6 receptor (IL-6R), suppressor of cytokine signaling 3 (SOCS3), phospho-STAT3-Y705 and phospho-Smad3 in comparison to N-HLFs (p Conclusions IPF-HLF paracrine signaling leads to IL-6R overexpression, which in turn, affects N-HLF survival. The IL-6/STAT3/Smad3 axis facilitates cellular responses that could potentially promote fibrotic disease. This interplay was successfully blocked by TCZ.

Vascular smooth muscle cells (VSMC) are highly specialized, and exhibit a contractile phenotype when mature and fully differentiated, being responsible for vessel homeostasis and blood pressure control. In response to pro-atherogenic stimuli VSMC alter their state of differentiation, increase proliferation and migration, resulting in SMC phenotypes ranging from contractile to synthetic. This variability is observed in cell morphology and expression level of marker genes for differentiation status. There is growing evidence that bone morphogenetic protein (BMP) signaling is involved in vascular diseases, including atherosclerosis. Here, we evaluated in vitro the role of specific agonists/antagonists belonging to the BMP pathway on dedifferentiation of VSMC harvested during early stages of atherosclerosis. RESULTS: Comparing primary VSMC isolated from aortas of susceptible ApoE-/- animals fed 8 weeks of western diet with their littermate controls fed usual diet, we observed that recombinant BMP4 was able to reduce SM22-alpha and alpha actin gene expression indicating dedifferentiation was under way. Unexpectedly, treatment with recombinant Gremlin-1, a known BMP antagonist, also reduced 4-6.5 folds gene expression of SM22-alpha, alpha-actin and, calponin, exclusively in VSMC from ApoE-/- animals, independently on the diet consumed. CONCLUSION: Our data show that BMP4 is capable of modulating of SM22-alpha and alpha actin gene expression, indicative of cell dedifferentiation in VSMC. Additionally, we report for first time that Gremlin-1 acts independently of the BMP pathway and selectively on VSMC from susceptible animals, reducing the expression of all genes evaluated.

Gremlin genes are known members of the DAN family of bone morphogenetic protein (BMP) antagonists, but their functions and regulatory mechanisms in ovarian follicular development of chicken remain unknown. The current study was designed to investigate the mRNA expression patterns of gremlin1 gene (GREM1) and its protein location in the follicles sampled, and to explore the biological effect of GREM1 on the prehierarchical follicular development. This work revealed that chicken GREM1 mRNA exhibits a constant expression level across all the prehierarchical follicles (PFs) from 1-4 mm to 7-8 mm in diameter, and the preovulatory follicles (from F6 to F1) by using RT-qPCR (P 0.05). The GREM1 protein is predominantly expressed in the oocytes and granulosa cells (GCs) of the PFs by immunohistochemistry. Furthermore, our data demonstrated that siRNA-mediated knockdown of GREM1 in the GCs resulted in a significant reduction in cell proliferation (P 0.001); conversely, overexpression of GREM1 in the GCs led to a remarkable increase in cell proliferation (P 0.001). Interestingly, the expression levels of proliferating cell nuclear antigen (PCNA) and cyclin D2 (CCND2) mRNA and proteins were notably increased when GREM1 expression was upregulated in the GCs (P 0.01), however, the expression levels of CYP11A1 and StAR were markedly downregulated (P 0.01). The current results showed that GREM1 gene plays a stimulatory role in GC proliferation during growth and development of the prehierarchical follicles in vitro but an inhibitory role in GC differentiation and steroidogenesis of the hen ovary follicles.