Bone Morphogenetic Protein Antagonist Research Articles

Black Phosphorus-Based Dynamic Self-Healing Hydrogel to Integrate Demineralized Bone Matrix and Noggin-Targeting siRNA for Synergistic Osteogenesis.

Although a demineralized bone matrix (DBM) is often used as an alternative to an autologous bone graft, its clinical application is still hampered by easy dispersion of DBM particles and insufficient osteoinductivity in the defect site. Herein, we designed a self-healing hydrogel for DBM that can rapidly restore its structural integrity after damage based on amino-rich black phosphorus (BP) nanosheets and aldehyde-functionalized hyaluronic acid (AHA). Given the increased expression of bone morphogenetic protein (BMP) antagonists by DBM stimulation, the osteogenic potency of DBM in the hydrogel carrier was further enhanced by abrogating the BMP antagonism. The BP/AHA hydrogel provided dynamic polymer-nanosheet networks that combine injectability, modability, and physical stability with high DBM loading, where the BP nanosheets served as osteogenic cross-linkers to promote biomineralization and deliver siRNA to suppress undesirable expression of BMP antagonist noggin by DBM. As a result, the BP/AHA hydrogel integrated with DBM and noggin-targeting siRNA synergistically promoted osteogenic differentiation of mesenchymal stem cells by enhancing BMP/Smad signaling. This work demonstrates a promising strategy to improve the efficacy of bone regeneration using bone graft.

Characterization of erythroferrone oligomerization and its impact on BMP antagonism

Hepcidin, a peptide hormone that negatively regulates iron metabolism, is expressed by bone morphogenetic protein (BMP) signaling. Erythroferrone (ERFE) is an extracellular protein that binds and inhibits BMP ligands, thus positively regulating iron import by indirectly suppressing hepcidin. This allows for rapid erythrocyte regeneration after blood loss. ERFE belongs to the C1Q/TNF related protein (CTRP) family and is suggested to adopt multiple oligomeric forms: a trimer, a hexamer, and a high molecular weight species. The molecular basis for how ERFE binds BMP ligands and how the different oligomeric states impact BMP inhibition are poorly understood. In this study, we demonstrated that ERFE activity is dependent on the presence of stable dimeric or trimeric ERFE, and that larger species are dispensable for BMP inhibition. Additionally, we used an in-silico approach to identify a helix, termed the ligand binding domain (LBD), that was predicted to bind BMPs and occlude the type I receptor pocket. We provide evidence that the LBD is crucial for activity through luciferase assays and surface plasmon resonance (SPR) analysis. Our findings provide new insight into how ERFE oligomerization impacts BMP inhibition, while identifying critical molecular features of ERFE essential for binding BMP ligands.

Склеростин-залежні механізми формування остеопорозу у хворих цукровим діабетом 2 типу та ожирінням

при обстеженні 103 пацієнтів на цукровий діабет 2 типу (ЦД), що у 83 випадкахперебігав на тлі збільшеної маси тіла або ожиріння, було встановлено вірогідне збільшеннявмісту склеростину у сироватці крові, яке мало пряму кореляційну залежність з індексоммаси тіла. Висловлено припущення, що зміни даного глікопротеїну, який входить до групиантагоністів кісткових морфогенетичних білків, відбуваються внаслідок багатьох процесів ворганізмі: збільшення синтезу ФНП-α адипоцитами, ендотелієм судин, клітинами нейроглії натлі пре- та менопаузи, що сприяє порушенню мінеральної щільності кісткової тканини, тобтоформуванню остеопорозу. Ендокринний остеопороз становить найбільшу групу вторинногоостеопорозу, провідною причиною якого є цукровий діабет 2 типу. В разі наявності ЦД 2 типу,який перебігає на тлі ожиріння, формуються умови до розвитку остеопоротичних станів, одниміз механізмів розвитку яких може виступати глікопротеїн склеростин. Метою дослідженнястало визначення структурно-функціонального стану кісткової тканини шляхом дослідженнявмісту склеростину у хворих на цукровий діабет 2 типу, що перебігає на тлі топографічнихособливостей розподілу жирової тканини. До роботи було залучено 103 хворих на ЦД 2 типу,який у 83 випадках перебігав в поєднанні з надлишковою вагою або ожирінням (основна група). Догрупи порівняння увійшло 20 хворих на ЦД 2 типу, що був діагностований у пацієнтів з незмінениміндексом маси тіла (ІМТ). Вік хворих коливався від 31 до 55 років та в середньому по групах складав43±4,6 років та 44,1±2,1 роки відповідно. В обох групах хворих переважали чоловіки 44 (53%) та11 (55%) осіб. Тривалість захворювання реєструвалась в межах від 1 до 13 років. Визначеннярозподілу жирової тканини в організмі досліджували за допомогою ваг для характеристикискладу тіла - OMRON BF511 (Японія). Ідентифікували вміст вісцеральної жирової тканини(ВЖТ) та підшкірної жирової тканини (ПЖТ). Активність склеростину в сироватці крові
 визначали імуноферментним методом з використанням комерційних тест-систем «Biomedi-ca» (США) згідно інструкції на імуноферментному аналізаторі «Labline-90» (Австрія). Обробку
 отриманих даних здійснювали методом варіаційної статистики за допомогою ліцензійногопрограмного забезпечення Stata 10 («StatSoft Inc.», США). Перевірка на нормальність розподілуданих виконувалася за допомогою критерію Шапіро-Уілка. Для параметрів з нормальнимрозподілом розраховували середню величину (M) ± стандартну помилку середньої (m). Отриманірезультати розглядалися як статистично значущі при p<0,05. В результаті було визначено, щозбільшення ІМТ супроводжувалось підвищенням показника склеростину. Тобто, виявлені зміниглікопротеїну, який входить до групи антагоністів кісткових морфогенетичних білків, дають змогу припустити порушення процесу диференціації остеобластів. Тобто, як ЦД 2 типу, такі кількісна зміна жирової тканини з її перерозподілом, негативно впливають на метаболізмкісткової тканини. Так, перебіг цукрового діабету супроводжується збільшенням активностіпрозапальних цитокінів, а саме, ФНП-α – складового цитокінового каскаду жирової тканини.Цей феномен також пов’язують із залученням до процесу судин (ендотеліальні клітинипродукують даний цитокін), нервової тканини (залучення клітин нейроглії) та метаболічнимизсувами в організмі. Окрім того F.Xu та співавт. в експерименті доведено, що остеокластогенезпригнічується при гіперглікемічних та гіперінсулінемічних станах. Тобто, у хворих на ЦД призбільшенні ІМТ складається негативний «тандем» через додаткове надходження з багатьохклітин ФНП-α та збільшення синтезу склеростину, що не тільки «запускає» процес порушеннямінеральної щільності кісткової тканини (МЩКТ), але й підтримує його впродовж всьогоперебігу захворювання. Також певна роль відводиться віковому аспекту обстежених хворихна ЦД (середній вік менопаузи у здорових жінок нашої країни становить 45 років), що такожсприяє пригніченню кісткового метаболізму. В такому разі збільшення активності склеростинув поєднанні з негативним фоном (ожиріння, період менопаузи) можна розглядати як один ізмеханізмів формування остеопоротичних змін. При цьому поєднуються механізми первинного тавторинного ОП, що посилює і прискорює процес порушення архітектоніки кістки. Тому можназробити висновки, що у хворих на цукровий діабет 2 типу у поєднанні з ожирінням виникаютьумови до зниження якості кісткової тканини через уповільнення кісткового метаболізму, щосприяє формуванню остеопоротичних станів.

The inhibitory effect of Gremlin-2 on adipogenesis suppresses breast cancer cell growth and metastasis

BackgroundGremlin-1 (GREM1) and Gremlin-2 (GREM2) are bone morphogenetic protein antagonists that play important roles in organogenesis, tissue differentiation, and tissue homeostasis. Although GREM1 has been reported to be involved in promoting various cancers, little has been reported about effects of GREM2 on cancer. Recently, it has been reported that GREM2 can inhibit adipogenesis in adipose-derived stromal/stem cells. However, as an inhibitor of adipogenesis, the role of GREM2 in cancer progression is not well understood yet.MethodsPre-adipocyte 3T3-L1 cells overexpressing mock or Grem2 were established using a lentiviral transduction system and differentiated into adipocytes-mock and adipocytes-Grem2, respectively. To investigate the effect of adipocyte-Grem2 on breast cancer cells, we analyzed the proliferative and invasion abilities of spheroids using a 3D co-culture system of breast cancer cells and adipocytes or conditioned medium (CM) of adipocytes. An orthotopic breast cancer mouse model was used to examine the role of adipocytes-Grem2 in breast cancer progression.ResultsGrem2 overexpression suppressed adipogenesis of 3T3-L1 cells. Proliferative and invasion abilities of spheroids formed by co-culturing MTV/TM-011 breast cancer cells and adipocytes-Grem2 were significantly reduced compared to those of spheroids formed by co-culturing MTV/TM-011 cells and adipocytes-mock. Compared to adipocytes-mock, adipocytes-Grem2 showed decreased mRNA expression of several adipokines, notably IL-6. The concentration of IL-6 in the CM of these cells was also decreased. Proliferative and invasive abilities of breast cancer cells reduced by adipocytes-Grem2 were restored by IL-6 treatment. Expression levels of vimentin, slug, and twist1 in breast cancer cells were decreased by treatment with CM of adipocytes-Grem2 but increased by IL-6 treatment. In orthotopic breast cancer mouse model, mice injected with both MTV/TM-011 cells and adipocytes-Grem2 showed smaller primary tumors and lower lung metastasis than controls. However, IL-6 administration increased both the size of primary tumor and the number of metastatic lung lesions, which were reduced by adipocytes-Grem2.ConclusionsOur study suggests that GREM2 overexpression in adipocytes can inhibit adipogenesis, reduce the expression and secretion of several adipokines, including IL-6, and ultimately inhibit breast cancer progression.

NBL1 Reduces Corneal Fibrosis and Scar Formation after Wounding.

Corneal scarring is a leading cause of blindness. Currently, there is no treatment to prevent and/or reduce corneal scar formation under pathological conditions. Our previous data showed that the NBL1 protein, also termed the DAN Family BMP (Bone morphogenetic protein) Antagonist, was highly expressed in corneal stromal cells upon wounding. Here, we examined the function of NBL1 in corneal wound healing. Mouse corneas were mechanically wounded, followed by a 2-week treatment using NBL1. Wounded corneas treated with vehicle or an Fc tag served as controls. Compared with the controls, NBL1 treatment facilitated wound re-epithelialization, partially restored the stromal thickness, and significantly reduced corneal scar formation. NBL1 treatment did not decrease immune cell infiltration, indicating that the anti-scarring effect was not dependent on immune suppression. We further examined the anti-fibrotic effect of NBL1 on human corneas. Pairs of human corneas were induced to form myofibroblasts (a key player in fibrosis and scarring) upon wounding and incubation in a medium containing TGF-β1. The OS corneas were treated with Fc as a control, and the OD corneas were treated with NBL1. Compared with the control, human corneas treated with NBL1 had significantly fewer myofibroblasts, which was consistent with these mouse data. A further study revealed that NBL1 treatment inhibited BMP canonical (phospho-Smad1/5) and no-canonical (phospho-p38) pathways in human corneas. Data show that NBL1 reduced corneal fibrosis and scar formation in mice and cultured human corneas. The underlying molecular mechanism is not certain because both anti-fibrotic Smad1/5 and pro-fibrotic p38 pathways were inhibited upon NBL1 treatment. Whether the p38 pathway dominates the Smad1/5 pathway during corneal fibrosis, leading to the anti-fibrotic effect of NBL1, needs further investigation.

Complementary modulation of BMP signaling improves bone healing efficiency

The bone morphogenetic protein (BMP) signaling pathway plays a crucial role in bone development and regeneration. While BMP-2 is widely used as an alternative to autograft, its clinical application has raised concerns about adverse side effects and deteriorated bone quality. Therefore, there is a need to develop more sophisticated approaches to regulate BMP signaling and promote bone regeneration. Here, we present a novel complementary strategy that targets both BMP antagonist noggin and agonist Trb3 to enhance bone defect repair without the application of exogenous BMP-2. In vitro studies showed that overexpression of Trb3 with simultaneous noggin suppression significantly promotes osteogenic differentiation of mesenchymal stem cells. This was accompanied by increased BMP/Smad signaling. We also developed sterosome nanocarriers, a non-phospholipid liposomal system, to achieve non-viral mediated noggin suppression and Trb3 overexpression. The gene-loaded sterosomes were integrated onto an apatite-coated polymer scaffold for in vivo calvarial defect implantation, resulting in robust bone healing compared to BMP-2 treatments. Our work provides a promising alternative for high-quality bone formation by regulating expression of BMP agonists and antagonists.

MiR-9-1 controls osteoblastic regulation of lymphopoiesis.

The highly conserved MicroRNA-9 (miR-9) family consists of three members. We discovered that miR-9-1 deletion reduced mature miR-9 expression, causing 43% of the mice to display smaller size and postweaning lethality. MiR-9-1-deficient mice with growth defects experienced severe lymphopenia, but other blood cells were unaffected. The lymphopenia wasn't due to defects in hematopoietic progenitors, as mutant bone marrow (BM) cells underwent normal lymphopoiesis after transplantation into wild-type recipients. Additionally, miR-9-1-deficient mice exhibited impaired osteoblastic bone formation, as mutant mesenchymal stem cells (MSCs) failed to differentiate into osteoblastic cells (OBs). RNA sequencing revealed reduced expression of master transcription factors for osteoblastic differentiation, Runt-related transcription factor 2 (Runx2) and Osterix (Osx), and genes related to collagen formation, extracellular matrix organization, and cell adhesion, in miR-9-1-deficient MSCs. Follistatin (Fst), an antagonist of bone morphogenetic proteins (BMPs), was found to be a direct target of miR-9-1. Its deficiency led to the up-regulation of Fst, inhibiting BMP signaling in MSCs, and reducing IL-7 and IGF-1. Thus, miR-9-1 controls osteoblastic regulation of lymphopoiesis by targeting the Fst/BMP/Smad signaling axis.

A ROCK inhibitor suppresses the transforming growth factor-beta-2-induced endothelial–mesenchymal transition in Schlemm’s canal endothelial cells

In the normal eye, most of the aqueous humor drains through the trabecular meshwork (TM) and Schlemm’s canal (SC). The concentration of transforming growth factor beta 2 (TGF-β2) is increased in the aqueous humor of primary open angle glaucoma patients. TGF-β2 increases outflow resistance by affecting the TM and SC, and endothelial–mesenchymal transition (EndMT) of SC cells is involved in these changes. Here, we investigated the effect of a ROCK inhibitor on TGF-β2-induced EndMT in SC cells. The ROCK inhibitor Y-27632 suppressed the TGF-β2-induced increase in the trans-endothelial electrical resistance (TER) and proliferation of SC cells. Y-27632 suppressed the expression of α-SMA, N-cadherin, and Snail, which are upregulated by TGF-β2. Moreover, TGF-β2 decreased mRNA levels of bone morphogenetic protein (BMP) 4 and increased those of the BMP antagonist gremlin (GREM1), but Y-27632 significantly suppressed these changes. Y-27632 also inhibited TGF-β2-induced phosphorylation of p-38 mitogen-activated protein kinase (MAPK). BMP4 and the p-38 MAPK inhibitor SB203580 suppressed the TGF-β2-induced TER elevation in SC cells. Moreover, SB203580 suppressed TGF-β2-induced upregulation of fibronectin, Snail, and GREM1. These results indicate that a ROCK inhibitor inhibited the TGF-β2-induced EndMT in SC cells, implying the involvement of p38 MAPK and BMP4 signaling.

Holoprosencephaly with a Special Form of Anophthalmia Result from Experimental Induction of bmp4, Oversaturating BMP Antagonists in Zebrafish.

Vision is likely our most prominent sense and a correct development of the eye is at its basis. Early eye development is tightly connected to the development of the forebrain. A single eye field and the prospective telencephalon are situated within the anterior neural plate (ANP). During normal development, both domains are split and consecutively, two optic vesicles and two telencephalic lobes emerge. If this process is hampered, the domains remain condensed at the midline. The resulting developmental disorder is termed holoprosencephaly (HPE). The typical ocular finding associated with intense forms of HPE is cyclopia. However, also anophthalmia and coloboma can be associated with HPE. Here, we report that a correct balance of Bone morphogenetic proteins (BMPs) and their antagonists are important for forebrain and eye field cleavage. Experimental induction of a BMP ligand results in a severe form of HPE showing anophthalmia. We identified a dysmorphic forebrain containing retinal progenitors, which we termed crypt-oculoid. Optic vesicle evagination is impaired due to a loss of rx3 and, consecutively, of cxcr4a. Our data further suggest that the subduction of prospective hypothalamic cells during neurulation and neural keel formation is affected by the induction of a BMP ligand.

Overexpression of GREM1 Improves the Survival Capacity of Aged Cardiac Mesenchymal Progenitor Cells via Upregulation of the ERK/NRF2-Associated Antioxidant Signal Pathway.

Ischemic heart disease is the leading cause of mortality in the United States. Progenitor cell therapy can restore myocardial structure and function. However, its efficacy is severely limited by cell aging and senescence. Gremlin-1 (GREM1), a member of the bone morphogenetic protein antagonist family, has been implicated in cell proliferation and survival. However, GREM1's role in cell aging and senescence has never been investigated in human cardiac mesenchymal progenitor cells (hMPCs). Therefore, this study assessed the hypothesis that overexpression of GREM1 rejuvenates the cardiac regenerative potential of aging hMPCs to a youthful stage and therefore allows better capacity for myocardial repair. We recently reported that a subpopulation of hMPCs with low mitochondrial membrane potential can be sorted from right atrial appendage-derived cells in patients with cardiomyopathy and exhibit cardiac reparative capacity in a mouse model of myocardial infarction. In this study, lentiviral particles were used to overexpress GREM1 in these hMPCs. Protein and mRNA expression were assessed through Western blot and RT-qPCR. FACS analysis for Annexin V/PI staining and lactate dehydrogenase assay were used to assess cell survival. It was observed that cell aging and cell senescence led to a decrease in GREM1 expression. In addition, overexpression of GREM1 led to a decrease in expression of senescence genes. Overexpression of GREM1 led to no significant change in cell proliferation. However, GREM1 appeared to have an anti-apoptotic effect, with an increase in survival and decrease in cytotoxicity evident in GREM1-overexpressing hMPCs. Overexpressing GREM1 also induced cytoprotective properties by decreasing reactive oxidative species and mitochondrial membrane potential. This result was associated with increased expression of antioxidant proteins, such as SOD1 and catalase, and activation of the ERK/NRF2 survival signal pathway. Inhibition of ERK led to a decrease in GREM1-mediated rejuvenation in terms of cell survival, which suggests that an ERK-dependent pathway may be involved. Taken altogether, these results indicate that overexpression of GREM1 can allow aging hMPCs to adopt a more robust phenotype with improved survival capacity, which is associated with an activated ERK/NRF2 antioxidant signal pathway.

Evaluation of GREM1 and THBS2 as prognostic markers in in non-small cell lung cancer.

To analyze the correlation between bone morphogenetic protein (BMP) antagonist Gremlin 1 (GREM1), Thrombospondin-2 (THBS2) and immune cell infiltration in non-small cell lung cancer (NSCLC) and the related clinical significance. A total of 150 NSCLC patients admitted to our hospital during May 2019-January 2022 were picked. The expression of GREM1 and THBS2 and the infiltration of immune cells in tumor tissues were detected through immunohistochemistry (IHC). These objects were graded as GREM1-positive group (n = 97), GREM1-negative group (n = 53), THBS2-positive group (n = 102) and THBS2-negative group (n = 48) according to the expression of GREM1 and THBS2. The correlation between the expression of GREM1 and THBS2 with immune cell infiltration and clinicopathological indicators was analyzed. Kaplan-Meier survival analysis was adopted to analyze the relationship between the expression of GREM1 and THBS2 and the prognosis in NSCLC tissues. The overall progression-free survival (PFS) of the two groups were compared by log-rank test. The results of IHC showed that the positive expression rate of GREM1 was 64.67% (97/150) in cancer tissues and 36.00% (54/150) in adjacent tissues. The positive expression rate of THBS2 was 68.00% (102/150) in cancer tissues and 25.33% (38/150) in adjacent tissues. The positive expression rate of GREM1 and THBS2 in cancer tissues was both much higher than that in adjacent tissues (P < 0.01). GREM1-positive group had much higher proportion of tumor diameter ≥ 2cm, stage III-IV and lymph-node metastasis than GREM1-negative group (P < 0.05). THBS2-positive group had markedly higher proportion of tumor diameter ≥ 2cm, stage III-IV, lymph-node metastasis and high differentiation than THBS2-negative group (P < 0.01). GREM1-positive group had much higher level of CD3 + T, and sharply lower level of CD8 + T and CD11c + DCs than GREM1-negative group (P < 0.01). THBS2-positive group had much higher level of CD3 + T, and sharply lower level of CD8 + T and CD11c + DCs than THBS2-negative group (P < 0.01). According to Kaplan-Meier survival analysis, the overall median progression-free survival (PFS) was 7.45months. Log-rank test showed that NSCLC patients with positive GREM1 and THBS2 had much shorter PFS than negative patients (P < 0.01). Log-rank test showed that the expression of GREM1 and THBS2 was related to the PFS of patients (P < 0.01). GREM1 and THBS2 were highly expressed in NSCLC tissues and acted as pro-oncogenes in the development and progression of NSCLC, which aggravated the disease by mediating immune cell infiltration.

Abstract P6-14-03: Oestrogen represses Noggin expression by interfering BMP/Smad signalling in ER positive breast cancer cells

Abstract Background Bone morphogenetic proteins (BMPs) are members of Transforming Growth Factor β (TGF-β) superfamily. BMPs are actively involved in the disease progression and bone metastasis of breast cancer. As a natural antagonist of BMP, Noggin plays important roles in the regulation of BMP signalling. It can facilitate spread of breast cancer cells to bone and subsequent colonisation and formation of osteolytic bone lesions. The present study aimed to investigate the regulatory mechanism for Noggin expression in oestrogen receptor (ER) positive breast cancer cells. Method Noggin expression was analysed in The Cancer Genome Atlas (TCGA ER (+) n=763, ER (-) n=216) and E-MTAB-6703 (ER (+) n=733, ER (-) n=421) cohorts. Correlation between Noggin and ER was evaluated using Spearman test. The expression of Noggin in an ER positive breast cancer cell line MCF-7 was determined by depriving the cells from oestrogen using phenol red-free DMEM supplemented with 10% charcoal stripped foetal calf serum or adding 10-10M 17-β-oestradiol. Activation of Smad-1/5/8 and involvement of BMP receptors in the oestrogen repressed Noggin expression were further examined using recombinant human BMP7 and a BMP receptor inhibitor (LDN-193189). Influence of Noggin on cellular functions was evaluated in MCF-7 cells with Noggin overexpression using a lentiviral Noggin expression vector. Results Noggin expression was negatively correlated with ERα in both TCGA BRCA (r=-0.162, p&amp;lt;0.01, n=1093) and E-MTAB-6703 (r=-0.078, p&amp;lt;0.01, n=2302) cohorts. The expression of Noggin was increased in the MCF-7 cells upon a deprivation from oestrogen which was further validated by adding 17-β-oestradiol. This is in line with the increased expression of Noggin observed in the MCF-7 cells with ER silencing (GSE27473). Furthermore, an increased level of phosphorylated Smad1/5/8 was seen in the MCF-7 being hungered from oestrogen which was prevented by adding 17-β-oestradiol and LDN-193189, respectively. As a result, the oestrogen hunger induced Noggin expression was also decreased by adding with 17-β-oestradiol and LDN-193189. To further investigate the influence of oestrogen on BMP-Smad signalling regulated Noggin expression, Noggin expression and phosphorylation of Smad1/5/8 and Smad3 were determined in MCF-7 cells which were treated with rhBMP7 and in combination with 17-β-oestradiol and LDN-193189, respectively. BMP7 induced Noggin expression and activation of Smad1/5/8 can be diminished by 17-β-oestradiol and LDN-193189. Noggin overexpression in MCF-7 cells resulted in an increase of proliferation. Conclusion Noggin expression can be repressed by oestrogen through an inference of the BMP- Smad signalling. Overexpression of Noggin promoted proliferation of MCF-7 cells. Further investigation is required to clarify the exact role of Noggin in ER positive breast cancer and its implication in the disease progression and current therapies. Citation Format: Ming Liu, Lin Ye, Wen G. Jiang. Oestrogen represses Noggin expression by interfering BMP/Smad signalling in ER positive breast cancer cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-03.

Chordin Like-1 Regulates Osteoblast and Adipocyte Differentiation Through Stabilizing Insulin-Like Growth Factor Binding Protein 3.

Chordin like-1 (CHRDL1) is an antagonist of bone morphogenetic proteins (BMPs) that acts through binding BMPs and blocking their interaction with BMP receptors. CHRDL1 plays a role in osteoblast differentiation but controversial effects were reported. On the other hand, the role of CHRDL1 in adipogenesis is unknown. In the present study, we investigated the function of CHRDL1 in regulating differentiation of osteoblasts and adipocytes and elucidated the underlying mechanism. CHRDL1 expression was downregulated during osteogenesis while it was upregulated during adipogenesis in primary cultured and established mesenchymal progenitor cell lines. Functional experiments revealed that CHRDL1 suppressed osteoblast differentiation and promoted adipocyte differentiation. Mechanistic explorations revealed that CHRDL1 is directly bound to insulin-like growth factor binding protein 3 (IGFBP3) and attenuated the degradation of the latter. Furthermore, CHRDL1 and IGFBP3 suppressed the activity of insulin receptor substrate 1 (IRS1)/AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin kinase complex 1 (mTORC1) signaling in progenitor cells undergoing osteogenic differentiation. By contrast, they activated AKT/mTORC1 signaling independently of IRS1 during adipogenic differentiation. CHRDL1 enhanced the interaction of nuclear IGFBP3 and retinoid X receptor α (RXRα) during adipogenesis, and inhibition of RXR inactivated AKT and attenuated the stimulation of adipogenic differentiation by CHRDL1. Overexpression of IGFBP3 relieved the perturbation of osteogenic and adipogenic differentiation of progenitor cells induced by CHRDL1 silencing. Finally, CHRDL1 and IGFBP3 were upregulated in the trabecular bone of aged mice. Our study provides evidence that CHRDL1 reciprocally regulates osteoblast and adipocyte differentiation through stabilizing IGFBP3 and differentially modulating AKT/mTORC1 signaling.

GREM1 in HER2+ breast cancer: worse prognosis, larger tumours and more bone metastasis

Breast cancer is the leading cause of female cancer deaths. Bone is the commonest site of breast cancer metastasis (40%) will be in the bone. Bone Morphogenetic Protein (BMP) antagonists have been shown to stimulate glycolysis as well as epithelial mesenchymal transition (EMT), which increases the motility of malignant cells and contributes to the development of metastases. The role of GREM1 in HER2+ breast cancer progression remains an under-researched yet promising area of study.

Integrated in Silico–in Vitro Rational Design of Osteogenic Peptides derived from the Armpit Epitope of Human Bone Morphogenetic Proteins

Bone morphogenetic proteins (BMPs) are multi-functional growth factors that initiate, promote and maintain cartilage and bone morphogenesis, differentiation and regeneration in both the developing embryo and adult. The proteins have a conformational wrist epitope and a linear knuckle epitope responsible for, respectively, type-I and type-II receptor binding, as well as a hybrid armpit epitope targeted by natural BMP antagonists. In this study, the recognition and interaction between human BMPs and their pan-antagonist Crossveinless was investigated systematically at molecular level. It is revealed that the armpit epitope shares a roughly common region over different BMPs, which consists of a loop segment and a turn segment that are sequentially discontinuous but spatially vicinal on these BMP protein surfaces. Turn segment is the primary binding site that can be bound effectively by Crossveinless using a tightly packed mode. The segment was further extended at its two termini to cover a complete double-stranded sheet of BMPs, which was then split from the interfacial context of BMP–Crossveinless complexes to derive a series of osteogenic peptides; they exhibit moderate intrinsic disorder in free state, but can be constrained into a native-like conformation by stapling a disulfide bridge across two strands of the sheet. The disulfide bridge was rationally designed and optimized to avoid disrupting the native interaction of BMP sheet peptides with the active pocket of Crossveinless. Biophysical assays substantiated that the binding affinities of resulting cyclic peptides were improved by 2–6-fold relative to their linear counterpart upon the stapling, in which the cyclic peptide Bmp7-sb1 (S[CLYFDDNSNVILC]K) derived from the double-stranded sheet region of BMP7 armpit epitope was determined to have the highest affinity to Crossveinless in all tested samples. These rationally designed epitope-derived peptides can be used as osteogenic agents to activate the human BMP signaling by competitively targeting their natural antagonist.

Dual SMAD inhibition enhances the longevity of human epididymis epithelial cells.

Primary human epididymis epithelial (HEE) cells are valuable reagents for functional studies on the human epididymis. We used them previously to determine the transcriptional networks that establish cell identity along the length of the epididymis from caput, corpus, and cauda. These studies on HEE cells and organoids derived from them revealed important cellular properties. However, similar to other primary cells, HEE cells undergo replicative senescence and de-differentiation in culture. A cocktail of small molecules was shown elsewhere to extend longevity of epithelial basal cells. The components included transforming growth factor β (TGF-β)/bone morphogenetic protein (BMP) antagonists, WNT agonist, and Rho-associated and coiled-coil containing protein kinase (ROCK) inhibitor (ROCKi), which together prevented the senescence-related upregulation of TGF-β signaling pathway members. Here, we treat HEE cells with the same cocktail and observed enhanced replicative potential and prolonged expression of markers of HEE differentiation. This treatment expands the differentiated HEE cell population available from individual epididymis tissue samples that can be used for molecular, cellular, and functional studies.

Canakinumab Effects on Erythropoiesis, Cardiovascular Risk, and Clonal Hematopoiesis: Proteogenomic Analysis of the Cantos Randomized Clinical Trial

Canakinumab is a monoclonal antibody that directly neutralizes interleukin (IL)-1β. In the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS) - a randomized, double-blind, placebo-controlled trial that enrolled 10,061 patients with previous myocardial infarction and high-sensitivity C-reactive protein (hsCRP) levels ≥2 mg/L - canakinumab prevented recurrent cardiovascular (CV) events (Ridker et al. NEJM 2017). Anemia of inflammation (AI) presents with mild to moderate anemia in the setting of systemic inflammatory conditions. The pathogenesis of AI involves deregulation of iron homeostasis via hepcidin and inflammatory cytokines such as IL-1β, IL-6, and TNF-α, resulting in skewed myeloid differentiation and decreased erythrocyte lifespan via enhanced erythrophagocytosis (Ganz et al. NEJM 2019). A CANTOS exploratory study revealed that canakinumab reduces the incidence of anemia and improves hemoglobin levels (Vallurupalli et al. Ann Intern Med. 2020). Among mutations associated with clonal hematopoiesis (CH), patients with TET2 variants had fewer major adverse CV events than those without CH mutations (Svensson et al. JAMA Cardiol. 2022). CH mutations in TET2 were associated with higher IL-1β levels (Bick et al. Nature 2020), generating the hypothesis that inhibition of IL-1β may provide preferential benefits to patients with CH mutations. To determine the association between CH mutations and clinical response to canakinumab and evaluate the underlying molecular mechanisms of anemia response, a proteogenomic study was performed in 13080 plasma samples from 4595 CANTOS participants, including highly multiplexed proteomics (4785 proteins) and targeted deep sequencing of CH mutations with variant allele fraction≥ 2% (Figure A). Longitudinal and cross-sectional analyses were performed in pre- and post-treatment samples from patients with at least one pre-treatment sample (baseline). CH mutations were associated with older age, anemia at baseline, and higher baseline IL-6 and TNF-α levels (all OR's >1.4 and p<0.003). Among patients without baseline anemia, canakinumab treatment prevented incidental anemia, compared to those treated with a placebo (without CH mutation: HR=0.72; 95% CI=0.61-0.84; P < 0.001, with CH mutations: HR=0.77; 95% CI=0.49-1.19; P =0.24). In individuals treated with canakinumab, however, those with CH mutations were more likely to develop incidental anemia compared to patients without CH mutations (HR=1.56; 95% CI=1.18-2.07; P=0.002). Conversely, among patients with baseline anemia, canakinumab treatment significantly improved hemoglobin levels in patients with CH mutations compared to a placebo (P< 0.001), whereas a significant difference was not found in hemoglobin levels of patients without CH mutations (Figure B). Enhanced hemoglobin response over time to canakinumab was observed in patients with concurrent baseline anemia and CH mutations, particularly in DNMT3A and TET2, compared to no CH mutations (all CH mutations, DNMT3A and TET2,P = 0.022, 0.004, 0.078 respectively). We observed that canakinumab treatment reversed multiple nodes of abnormal erythropoiesis associated with AI. Canakinumab treatment was associated with lower hepcidin and inflammatory cytokines (IL-6 and TNF-α) and down-regulation of pathways for complement activation and phagocytosis, which may prolong RBC lifespan and promote erythropoiesis. Higher odds of having both an increase in hemoglobin and a decrease in hepcidin (OR=1.88; 95% CI=1.62 - 2.17; P<0.001 ) and both an increase in hemoglobin and a decrease in IL-6 (OR=3.46; 95%CI=3.00 - 4.00; P<0.001) were observed with canakinumab treatment compared to placebo. The characterization of proteomic profiles in patients with CH mutations revealed novel biological links in dysregulation of RBC homeostasis, cardiovascular risk biomarkers, inhibitory immune molecules, and bone morphogenetic protein antagonists, which were reversed by canakinumab treatment more significantly in patients with TET2 mutations. Collectively this data provides direct clinical evidence that patients with concurrent anemia and CH mutations can benefit from canakinumab treatment by suppressing CH-mediated activation of the IL-1β/IL-6 axis and reversing ineffective erythropoiesis via multiple mechanisms, and the type of CH mutations may be associated with distinct clinical and biological response. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Multifaceted Functions of TWSG1: From Embryogenesis to Cancer Development.

Bone morphogenetic proteins (BMPs) play an important role in development. Twisted gastrulation BMP signaling modulator 1 (TWSG1) was initially identified as a regulator of the dorsoventral axis formation in Drosophila. The mechanism of BMP signaling modulation by TWSG1 is complex. TWSG1 inhibits BMP signaling by binding to BMP ligands including BMP4, whereas it enhances signaling by interacting with Chordin, a BMP antagonist. Therefore, TWSG1 can act as both a BMP agonist and antagonist. TWSG1 has various functions ranging from embryogenesis to cancer progression. TWSG1 knockout mice showed neural, craniofacial, and mammary defects. TWSG1 also regulated erythropoiesis and thymocyte development. Furthermore, the relationship between TWSG1 and cancer has been elucidated. Allelic loss of TWSG1 was detected in colorectal cancer. TWSG1 expression was upregulated in papillary thyroid carcinoma and glioblastoma but downregulated in gastric and endometrial cancers. TWSG1 suppressed BMP7-enhanced sphere formation and migration in endometrial cancer cells, indicating its tumor-suppressive role. Further studies are required to clarify the TWSG1 function and its association with BMP signaling in cancer development. Finally, TWSG1 is abundantly expressed in human and mouse ovaries and sustains follicular growth in rodent ovaries. Thus, TWSG1 has various functions ranging from fertility to cancer. Therefore, TWSG1 signaling modulation may be beneficial in treating specific diseases such as cancer.

The role of BMP4 in adipose-derived stem cell differentiation: A minireview.

Bone morphogenetic protein 4 (BMP4) is a member of the transforming growth factor beta (TGF-β) superfamily of cytokines responsible for stem cells' commitment to differentiation, proliferation, and maturation. To date, various studies have utilized BMP4 as a chemical inducer for in vitro differentiation of human mesenchymal stem cells (MSCs) based on its potential. BMP4 drives in vitro differentiation of ADSC via TGF-β signaling pathway by interactions with BMP receptors leading to the activation of smad-dependent and smad-independent pathways. The BMP4 signaling pathways are regulated by intracellular and extracellular BMP4 antagonists. Extracellular BMP4 antagonist prevents interaction between BMP4 ligand to its receptors, while intracellular BMP4 antagonist shutdowns the smad-dependent pathways through multiple mechanisms. BMP4 proved as one of the popular differentiation factors to induce ADSC differentiation into cell from mesodermal origin. However, addition of all-trans retinoic acid is also needed in trans-differentiation of ADSC into ectodermal lineage cells. Suggesting that both BMP4 and RA signaling pathways may be necessary to be activated for in vitro trans-differentiation of ADSC.

Fluvastatin promotes chondrogenic differentiation of adipose-derived mesenchymal stem cells by inducing bone morphogenetic protein 2

BackgroundAdipose-derived mesenchymal stem cells (ADMSCs) are a promising source of material source for medical regeneration of cartilage. Growth factors, including transforming growth factor-β (TGFβ) subfamily members and bone morphogenetic proteins (BMPs), play important roles in inducing and promoting chondrogenic differentiation of MSCs. However, these exogenous growth factors have some drawbacks related to their cost, biological half-life, and safety for clinical application. Several studies have reported that statins, the competitive inhibitors of 3-hydroxy-2-methylglutaryl coenzyme A (HMG-CoA) reductase, induce the expression of BMP2 in multiple cell types as the pleotropic effects. The objective of this study was to investigate the effects of fluvastatin during chondrogenic differentiation of human ADMSCs (hADMSCs).MethodsThe effects of fluvastatin were analyzed during chondrogenic differentiation of hADMSCs in the pellet culture without exogenous growth factors by qRT-PCR and histology. For functional studies, Noggin, an antagonist of BMPs, mevalonic acid (MVA) and geranylgeranyl pyrophosphate (GGPP), metabolites of the mevalonate pathway, ROCK inhibitor (Y27632), or RAC1 inhibitor (NSC23766) were applied to cells during chondrogenic differentiation. Furthermore, RhoA activity was measured by RhoA pulldown assay during chondrogenic differentiation with or without fluvastatin. Statistically significant differences between groups were determined by Student’s t-test or the Tukey–Kramer test.ResultsFluvastatin-treated cells expressed higher levels of BMP2, SOX9, ACAN, and COL2A1 than control cells, and accumulated higher levels of glycosaminoglycans (GAGs). Noggin significantly inhibited the fluvastatin-mediated upregulation of ACAN and COL2A1. Both MVA and GGPP suppressed the effects of fluvastatin on the expressions of BMP2, SOX9, ACAN, and COL2A1. Furthermore, fluvastatin suppressed the RhoA activity, and inhibition of RhoA–ROCK signaling by Y27632 increased the expressions of BMP2, SOX9, ACAN, and COL2A1, as well as fluvastatin.ConclusionsOur results suggest that fluvastatin promotes chondrogenic differentiation of hADMSCs by inducing endogenous BMP2, and that one of the mechanisms underlying the effects is inhibition of RhoA–ROCK signaling via suppression of GGPP. Fluvastatin is a safe and low-cost compound that holds promise for use in transplantation of hADMSCs for cartilage regeneration.