TGF-beta Family Members Research Articles

Abstract B072: SMAD4 loss allows progression from benign to malignant disease in intraductal papillary mucinous neoplasms

Abstract As increasing numbers of MRIs and other scans are ordered for unrelated diseases, more and more people are being diagnosed with cystic neoplasms of the pancreas. While many or most of these will likely remain benign, intraductal papillary mucinous neoplasms (IPMNs) have a risk of progression to cancer and IPMN is now recognized as another precursor to pancreatic ductal adenocarcinoma. We are investigating the mechanisms by which IPMNs progress from benign to malignant disease in order to identify markers of progression and targets to block that progression. We found that loss of SMAD4 in conjunction with Krasmutation leads to IPMN that quickly becomes invasive in a mouse model. Examining human IPMN tissues, we found that only one in 46 benign IPMN tumors lacked detectable SMAD4 protein. Among 19 cases of invasive IPMNs, the sites of active invasion were SMAD4 negative in 7 cases, nuclear negative/cytoplasm positive in 3 cases (suggesting no active canonical signaling), and a mix of positive and negative cells in 3 cases, implicating complete or partial loss of SMAD-mediated signaling in 68% of actively invading cells. In order to understand how SMAD signaling blocks progression to invasion, we are examining cell lines from a benign mouse model of IPMN. While these lines do not have endogenous TGF beta signaling, they are capable of responding to exogenous TGF beta, suggesting that TGF beta-related ligands may be supplied by the tumor microenvironment rather than the tumor cells themselves. We are currently determining how different TGF beta family members contribute to regulation of growth and invasion in these cells. In summary, loss of the tumor suppressor SMAD4 allows an otherwise benign disease to progress to invasive cancer. Citation Format: Anna L. Means, Hannah Yoo, Quoc-Huy Trinh, Roshini Thiagarajan, Jinghuan Zi, Frank L. Revetta, Marcus C. Tan, Kathleen E. DelGiorno, Mary K. Washington. SMAD4 loss allows progression from benign to malignant disease in intraductal papillary mucinous neoplasms [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B072.

Abstract P1119: Serum Gdf-15 is a Determinant Factor of Renal Vascular Resistance in Patients With Essential Hypertension

Introduction and Aims: Growth differentiation factor-15 (GDF-15), a member of TGF-beta family, has been implicated in several pathological conditions, which include inflammation, cancer, cardiovascular, pulmonary and renal diseases. Recently it is reported that serum GDF-15 may be a marker for chronic kidney disease (CKD) and CKD progression. In present study, we evaluated whether serum GDF-15 is associated with renal vascular resistance evaluated by resistive index (RI) measured in Ultrasound, which is the marker of renal injury. Methods: Eighty patients with essential hypertension were enrolled in this study. They were evaluated on serum GDF-15 and renal RI as well as for conventional atherosclerotic risk factors. Results: Mean age of all patients was 66 ± 12 years (35 males and 45 females). Mean eGFR and renal RI was 68.9 ± 23.0ml / min / 1.73m2 and 0.71 ± 0.07 respectively. Mean serum GDF-15 was 1492 ± 1319 pg/ml. RI correlated with GDF-15 (r=0.47, P<0.001), age (r=0.40, p<0.001), eGFR (r=-0.34, p=0.002) and diastolic BP (r=-0.56, p<0.001) in simple regression analysis. Multiple regression analysis showed GDF-15 (beta=0.31, P=0.005) and systolic BP (beta=0.35, P<0.001) and diastolic BP (beta=-0.74, P<0.001) were the independent determinants of renal RI. Conclusions: GDF-15 is associated with renal vascular resistance and may become a nephropathic marker in patients with hypertension.

Targeted Derivation of Organotypic Glucose- and GLP-1-Responsive β Cells Prior to Transplantation into Diabetic Recipients.

SummaryGeneration of functional β cells from pluripotent sources would accelerate diagnostic and therapeutic applications for diabetes research and therapy. However, it has been challenging to generate competent β cells with dynamic insulin-secretory capacity to glucose and incretin stimulations. We introduced transcription factors, critical for β-cell development and function, in differentiating human induced pluripotent stem cells (PSCs) and assessed the impact on the functionality of derived β-cell (psBC) progeny. A perifusion system revealed stepwise transduction of the PDX1, NEUROG3, and MAFA triad (PNM) enabled in vitro generation of psBCs with glucose and GLP-1 responsiveness within 3 weeks. PNM transduction upregulated genes associated with glucose sensing, insulin secretion, and β-cell maturation. In recipient diabetic mice, PNM-transduced psBCs showed glucose-responsive insulin secretion as early as 1 week post transplantation. Thus, enhanced pre-emptive β-cell specification of PSCs by PNM drives generation of glucose- and incretin-responsive psBCs in vitro, offering a competent tissue-primed biotherapy.

Growth differentiation factor (GDF-15) concentration combined with Ca125 levels in serum is superior to commonly used cancer biomarkers in differentiation of pancreatic mass.

Pancreatic cancer (PDAC) will have been the second leading cancer-related death in the United States by 2020, according to current estimation. Its late manifestation and the lack of good early detection methods are the cause of extremely low survival rates. Therefore, there is an urgent need to develop highly sensitive and specific marker. GDF-15, a member of TGFbeta family, has recently emerged as a protein playing an important role in carcinogenesis of various neoplasms. Our aim was to assess the potential of GDF-15, IL-17, IL-23 serum concentration, and the panel of PDAC markers in differentiating pancreatic adenocarcinoma from chronic pancreatitis. Sixty-three consecutive patients operated on due to pancreatobiliary lesions were enrolled in this study. Levels of CEA, CA125 and Ca19-9 were assessed using standard laboratory protocols. A sample of serum was collected prior to the surgery via central line. Levels of GDF-15, Il-17, Il-23 were measured using a ELISA kit. After standard pathological examination of specimens obtained on surgery, patients were divided into 2 groups: 42 patients with pancreatic adenocarcinoma and 21 patients with focal chronic pancreatitis. Mean GDF-15 concentration in patients with CP vs PDAC was 2247.95 (± 179.27) vs 7694.58 (± 1878.94) [pg/mL] respectively (p= 0.011). Mean concentration of Il-17, Il-23, Ca19-9, Ca125, Ca15-3, CEA in patients with CP and PDAC was 862.36 (± 30.84) vs 841.83 (± 33.94) p= 0.833; 127.85 (± 5.87) vs 127.51 (± 9.74) p= 0.175; 34.95 (± 23.34) vs 266.62 (±49.7) p= 0.001; 13.4 (± 1.6) vs 39.27 (± 6.85) p= 0.005; 18.4 (± 1.48) vs 20.2 (± 1.38) p= 0.416; 1.96 (± 0.38) vs 5.93 (± 1.74) p= 0.004 respectively. In order to compare these markers with the routinely used ones, ROC curve was built. CA19-9 with clinically used cut-off point of ⩾ 36 IU/mL has specificity of 90.5% and sensitivity of 57.14%. At the same time GDF-15 with the optimal cut-off point of 2.7 ng/mL has specificity of 76.19% and sensitivity of 73.8%. Although in our research group CA19-9 has an excellent specificity, its usefulness is hampered by its low sensitivity. On the other hand, GDF-15 parameters are well-balanced making it a more useful biomarker of PDAC. In conclusion, GDF-15 is more accurate than Ca19-9 in differentiating pancreatic mass due to chronic pancreatitis from pancreatic adenocarcinoma. Interleukin 17 and 23 cannot be considered as PDAC biomarkers. GDF-15 concentration in serum should be further investigated in order to assess their usefulness in pancreatic adenocarcinoma diagnosis.

Proteomics approach to identify novel metastatic bone markers from the secretome of PC-3 prostate cancer cells.

Prostate cancer is the leading type of cancer diagnosed, and the most frequent cause of worldwide male cancer-related deaths annually. The limitations of current prostate cancer screening tests demand the identification of novel biomarkers for the early diagnosis of prostate cancer bone metastasis. In the present study, we performed a proteomic analysis of secreted proteins from the prostate cancer bone metastasis cell line, PC-3, and the normal prostate cell line, RWPE-1. We thus quantified 917 proteins, of which 68 were found to be secreted at higher levels by PC-3 than by RWPE-1 cells via LC-MS/MS. To characterize the highly secreted proteins in the PC-3 cell line and thereby identify biomarker proteins, we divided the quantifiable proteins into four quantitative categories (Q1-Q4). The KEGG lysine degradation and osteoclast differentiation pathways were demonstrated to be enriched in the highly secreted Q4 protein group. Transforming growth factor (TGF) beta family proteins related to osteoclast differentiation were identified as key regulators of PC-3 cell proliferation. Immunoblotting was used to confirm the observed high level of pentraxin, follistatin, TGF-beta family members, and serpin B3 secretion by PC-3 cells. From the collective results of the present study, we suggest that serpin B3 is a promising novel biomarker candidate for the diagnosis of prostate cancer bone metastasis.

Exogenous GDF11 induces cardiac and skeletal muscle dysfunction and wasting.

Growth differentiation factor 11 (GDF11), a TGF-beta superfamily member, is highly homologous to myostatin and essential for embryonic patterning and organogenesis. Reports of GDF11 effects on adult tissues are conflicting, with some describing anti-aging and pro-regenerative activities on the heart and skeletal muscle while others opposite or no effects. Herein, we sought to determine the in vivo cardiac and skeletal muscle effects of excess GDF11. Mice were injected with GDF11 secreting cells, an identical model to that used to initially identify the in vivo effects of myostatin. GDF11 exposure in mice induced whole body wasting and profound loss of function in cardiac and skeletal muscle over a 14-day period. Loss of cardiac mass preceded skeletal muscle loss. Cardiac histologic and echocardiographic evaluation demonstrated loss of ventricular muscle wall thickness, decreased cardiomyocyte size, and decreased cardiac function 10days following initiation of GDF11 exposure. Changes in skeletal muscle after GDF11 exposure were manifest at day 13 and wereassociated with wasting, decreased fiber size, and reduced strength. Changes in cardiomyocytes and skeletal muscle fibers were associated with activation of SMAD2, the ubiquitin-proteasome pathway and autophagy. Thus,GDF11 over administration in vivo results in cardiac and skeletal muscle loss, dysfunction, and death. Here, serum levels of GDF11 by Western blotting were 1.5-fold increased over controls. Although GDF11 effects in vivo are likely dose, route, and duration dependent, its physiologic changes are similar to myostatin and other Activin receptors ligands. These data support that GDF11, like its other closely related TGF-beta family members, induces loss of cardiac and skeletal muscle mass and function.

Maternal messages to live by: a personal historical perspective.

In the 1980s, the study of localized maternal mRNAs was just emerging as a new research area. Classic embryological studies had linked the inheritance of cytoplasmic domains with specific cell lineages, but the underlying molecular nature of these putative determinants remained a mystery. The model system Xenopus would play a pivotal role in the progress of this new field. In fact, the first localized maternal mRNA to be identified and cloned from any organism was Xenopus vg1, a TGF-beta family member. This seminal finding opened the door to many subsequent studies focused on how RNAs are localized and what functions they had in development. As the field moves into the future, Xenopus remains the system of choice for studies identifying RNA/protein transport particles and maternal RNAs through RNA-sequencing.

Growth Differentiation Factor-15 (GDF-15) Is a New Biomarker with Independent Prognostic Significance for Survival and Renal Outcomes in Different Cohorts of Patients with Light Chain (AL) Amyloidosis

Growth differentiation factor-15 (GDF-15), a member of TGF-beta family, is involved in several pathological conditions, which include inflammation, cancer, cardiovascular, pulmonary and renal diseases. Serum GDF-15 levels add prognostic information to conventional prognostic factors, such as NT-proBNP and troponins, in cardiovascular disorders and also have shown to be associated with renal damage and risk of end stage renal disease in patients with diabetes. Based on the above data, we evaluated the prognostic value of GDF-15 levels in patients with AL amyloidosis and showed that in a cohort of 77 patients GDF-15 was associated with early death, shorter survival and progression to dialysis, independently of the cardiac biomarkers and renal stage. In order to validate the prognostic value of serum levels of GDF-15, we evaluated GDF-15 in two independent cohorts of patients treated in two different centers (Pavia Amyloidosis Center and Department of Clinical Therapeutics, Athens).Circulating levels of GDF-15 were measured by a novel pre-commercial immunoassay (Roche Diagnostics) in stored serum. The Pavia cohort included 202 and the Athens cohort included 107 patients with AL amyloidosis. Standard criteria were used for the diagnosis, evaluation of organ involvement and cardiobiomarker-based risk stratification. Renal staging was based on the system proposed by Palladini et al, based on baseline proteinuria >5 gr/day and eGFR <50 ml/min.Median age and involved FC (iFLC) levels were similar between the two cohorts but there were differences in other baseline characteristics including heart involvement (77% vs 62% for Pavia vs Athens, p=0.007), Mayo stage (15%, 43% & 42% for stage 1, 2 & 3 in Pavia cohort, vs 26%, 43% & 31% in Athens cohort, p=0.04, but stage 3B was similar, 22% vs 24%). Also there were differences in peripheral nerve involvement (11% in Pavia vs 25% in Athens cohort, p=0.001). Renal involvement (67% vs 72%, p=0.415), median eGFR and renal stage distribution were similar between the two cohorts (p=0.544). Median follow up for the Pavia cohort was 18 months and for the Athens cohort was 45 months (p<0.001). Survival at 2 years was 59% for Pavia and 56% for Athens cohort.Median GDF-15 levels was 3027 pg/ml in Pavia (range 624 to >100000 pg/ml) and 3854 pg/ml (range 626-71475 pg/ml) in Athens cohort (p=0.09); the upper quartile of GDF-15 levels however was ≥5658 pg/ml for the Pavia and ≥7553 pg/ml for Athens cohort, while 90% and 94% of patients in the two cohorts had GDF-15 levels >1200 pg/ml (the upper limit of normal for individuals without cardiovascular disease).We then evaluated the prognostic significance of GDF-15 levels in the two cohorts by applying the previously identified cutoff of 7575 pg/ml. GDF-15 above cutoff was associated with significantly shorter survival both in Pavia (17 months vs not reached, p=0.003) and in Athens cohort (13 vs 47 months, p=0.03) (Figure 1). A multivariate model that included Mayo stage, separately for each cohort was applied. In both cohorts GDF-15 retained independent prognostic significance over Mayo stage, having a hazard ratio (HR) of 1.9 (95% CI 1.2-3.3, p=0.01) in Pavia cohort (the respective HRs for Mayo stage -2 vs -1 was 2.1 and for stage -3 vs stage -1 was 4); in Athens cohort HR for GDF-15 was 1.8 (95% CI 1.2-3.6, p=0.03) and the respective HRs for Mayo stage -2 vs -1 was 5.3 and for stage 3- vs stage -1 was 6.8.We then evaluated the prognostic significance regarding renal outcomes (dialysis): GDF-15 >4000 pg/ml was associated with a HR of 6 (95% CI 2015.6, p=0.001) in Athens cohort (progression to dialysis within 2 years in 7% vs 47%); however, very few events have occurred in Pavia cohort and analysis was inconclusive. Although renal stage discriminated 3 groups in univariate analysis (p=0.03), in multivariate analysis, GDF-15 >4000 pg/ml outperformed renal stage and was the only independent prognostic factor for dialysis risk.In conclusion, our study validated and confirmed in two independent cohorts, with differences in their characteristics, the prognostic value of GDF-15, which emerges as a novel biomarker with prognostic implications for different outcomes in patients with AL amyloidosis. Importantly, GDF-15 emerges also as new biomarkers for renal outcomes in patients with AL amyloidosis. [Display omitted] [Display omitted] DisclosuresKastritis:Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Genesis: Consultancy, Honoraria. Merlini:Takeda and Janssen-Cilag: Honoraria. Terpos:Genesis: Consultancy, Honoraria, Other: Travel expenses; Janssen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Celgene: Honoraria; Takeda: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Novartis: Honoraria. Dimopoulos:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Palladini:Prothena: Honoraria.

Growth differentiation factor-15/macrophage inhibitory cytokine-1 induction after kidney and lung injury.

The immunoregulatory cytokine macrophage inhibitory cytokine-1 (MIC-1), a divergent TGF-beta family member, and its murine ortholog, growth/differentiation factor-15 (GDF-15) are induced in hepatocytes by surgical and chemical injury and heat shock. To better understand the in vivo role this factor plays in organ injury, we examined the regulation of GDF-15 in murine models of kidney and lung injury. We demonstrate herein induction of GDF-15/MIC-1 after surgical, toxic/genotoxic, ischemic, and hyperoxic kidney or lung injury. Gdf15 induction was independent of protein synthesis, a hallmark of immediate-early gene regulation. Although TNF induced GDF-15 expression, injury-elicited Gdf15 expression was not reduced in mice deficient for both TNF receptor subtype. Furthermore, although the stress sensor p53 is known to induce GDF-15/MIC-1 expression, injury-elicited Gdf15 expression was unchanged in p53-null mice. Our results demonstrate that GDF-15 induction after organ injury is a hallmark of many tissues. These data demonstrate that GDF-15/MIC-1 is an early mediator of the injury response in kidney and lung that might regulate inflammation, cell survival, proliferation, and apoptosis in a variety of injured tissues and disease processes.

Estrogen mediated regulation of TGFβ pathway signaling in cardiac hypertrophy (795.4)

It is well documented that pre‐menopausal women are protected from heart disease, in part through the effects of estrogen. It has been established that estrogen treatment prevents cardiomyocyte hypertrophy and that estrogen attenuates the prohypertrophy TGFBeta (TGFb) signaling family. However, it has yet to be established whether estrogen is in part mediating its cardioprotective effects via the silencing of TGFb signaling. To address the differential control of the TGFb family member expression in female vs. male mice in the setting of cardiac hypertrophy, we performed transaortic constriction (TAC) to induce hypertrophy. We then analyzed gene and miRNA expression for TGFb family members and potential targeting miRNAs, several of which are known to be induced by estrogen. Next Generation Sequencing was performed to establish miRNA profiles in male and female following the TAC. Differential expression of TGFb targeting miRNAs was confirmed by TaqMan assays. TaqMan also revealed that many of the TGFb family members were preferentially downregulated in female TAC mice as compared to age matched male TAC mice. To define the mechanisms by which estrogen regulates TGFb expression we are performing a series of estrogen receptor activation studies in an in vitro cardiomyocyte hypertrophy model system. Preliminary results show distinct activation of TGFb family members and targeting miRNAs in the traditional nuclear receptors. The direct role for estrogen‐induced miRNAs will be further investigated using antagomirs in vitro. These data taken together will define the underlying estrogen mediated pathways related to TGFb signaling and could lead to new therapeutic targets for heart disease.Grant Funding Source: Supported by an Institutional Development Award (IDeA) under grant number R15HL108192

Abstract A42: An albumin leader sequence coupled with a cleavage site modification enhances the yield of recombinant c-terminal Mullerian inhibiting substance

Abstract Mullerian Inhibiting Substance (MIS) has been shown to inhibit ovarian cancer cells both in-vitro and in-vivo. MIS inhibits a putative ovarian cancer progenitor cell population enriched by a panel of CD44+, CD24+, Ep-CAM+, and E-cadherin- cell surface markers. In order to accommodate clinical testing of MIS in ovarian cancer patients, the production of recombinant human MIS must be optimized to increase yield and purity. Here, we show that the substitution of the MIS leader sequence to that of human serum albumin (HSAL), combined with a modification of the endogenous cleavage site from RAQR/S to a furin/kex2 RARR/S consensus site results in high expression, increased c-terminus cleavage and a reduction in unwanted cryptic internal cleavage products when produced in CHO cells. Purified MIS containing the HSAL modification displays increased potency to induce regression of the Mullerian duct in fetal rat embryonic urogenital ridge assays, reflecting the increased cleavage. The modifications herein described, which may be applicable to other TGF-beta family members, will facilitate production of active recombinant c-terminal MIS for clinical trials in ovarian cancer. Citation Format: David Pepin, Mien Hoeng, Fotini Nicolaou, Katherine Hendren, Leo Benedict, Amanda Sosulski, Ahmad Al-Moujahed, Demetrious Vavvas, Patricia K. Donahoe. An albumin leader sequence coupled with a cleavage site modification enhances the yield of recombinant c-terminal Mullerian inhibiting substance. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A42.

Characterization of a novel missense mutation in the prodomain of GDF5, which underlies brachydactyly type C and mild Grebe type chondrodysplasia in a large Pakistani family

All TGF-beta family members have a prodomain that is important for secretion. Lack of secretion of a TGF-beta family member GDF5 is known to underlie some skeletal abnormalities, such as brachydactyly type C that is characterized by a huge and unexplained phenotypic variability. To search for potential phenotypic modifiers regulating secretion of GDF5, we compared cells overexpressing wild type (Wt) GDF5 and GDF5 with a novel mutation in the prodomain identified in a large Pakistani family with Brachydactyly type C and mild Grebe type chondrodyslplasia (c527T>C; p.Leu176Pro). Initial in vitro expression studies revealed that the p.Leu176Pro mutant (Mut) GDF5 was not secreted outside the cells. We subsequently showed that GDF5 was capable of forming a complex with latent transforming growth factor binding proteins, LTBP1 and LTBP2. Furthermore, secretion of LTBP1 and LTBP2 was severely impaired in cells expressing the Mut-GDF5 compared to Wt-GDF5. Finally, we demonstrated that secretion of Wt-GDF5 was inhibited by the Mut-GDF5, but only when LTBP (LTBP1 or LTBP2) was co-expressed. Based on these findings, we suggest a novel model, where the dosage of secretory co-factors or stabilizing proteins like LTBP1 and LTBP2 in the microenvironment may affect the extent of GDF5 secretion and thereby function as modifiers in phenotypes caused by GDF5 mutations.

The Chick Embryo as an Experimental System for Melanoma Cell Invasion

BackgroundA primary cutaneous melanoma will not kill the patient, but its metastases. Since in vitro studies on melanoma cells in 2-D cultures do often not reflect reality, 3-D models might come closer to the physiological situation in the patient during cancer initiation and progression.Methodology/Principal FindingsHere, we describe the chick embryo model for in vivo studies of melanoma cell migration and invasion. After transplantation of neural crest-derived melanoma cells into the neural tube, the melanoma cells resume neural crest cell migration along the medial and lateral pathways and finally undergo apoptosis in the target areas. Upon transplantation into ectopic areas such as the hindbrain or the optic cup malignant invasion and local tissue destruction occurs. In contrast, melanocytes are not able to spontaneously resume neural crest cell migration. However, malignant invasion can be induced in melanocytes by pre-treatment with the TGF-beta family members bone morphegenetic protein-2 or nodal. Transplantation of MCF7 breast cancer cells yields a different growth pattern in the rhombencephalon than melanoma cells.Conclusions/SignificanceThe chick embryo model is a feasible, cost-effective in vivo system to study invasion by cancer cells in an embryonic environment. It may be useful to study invasive behavior induced by embryonic oncogenes and for targeted manipulation of melanoma or breast cancer cells aiming at ablation of invasive properties.

TGF beta family members function in uterine healthy and fibrotic smooth muscle cells

Uterine leiomyomas are the most common benign tumors of fertile women and the most common indication for hysterectomy. Despite the high prevalence, significant health problems, and huge economical impact on the healthcare system, relatively little is understood about the etiology and pathophysiology of uterine leiomyoma (1). Consequently, medical treatments are still limited (2). The role of the growth factors as ultimate mediators of the steroids hormone is evident in the modulation of the cell proliferation and the morphological cells appearance (3). Activin-A and myostatin are growth factors belonging to TGF-β super family expressed and acting in myometrial (4,5) and leiomyoma cells (6) We aimed to explore the functions of activin and myostatin in human myometrial and leiomyoma cells. First we tested both Smad and non-Smad signaling pathways by western blot. We found that activin-A and myostatin can activate only Smad signaling pathway in both myometrial and leiomyoma cells. Next we explored the effect on cell proliferation and on fibrotic phenotype. We found that activin-A and myostatin are able to suppress primary myometrial cell proliferation but they cannot alter the proliferation of leiomyoma cells. In the next step, we found that activin-A can significantly increase fibronectin expression in leiomyoma cells. Those above results suggest that activin-A and myostatin may express antiproliferative and/or fibrotic effects depending on the cell types by activating Smad signaling pathway.

Cells derived from murine induced pluripotent stem cells (iPSC) by treatment with members of TGF-beta family give rise to osteoblasts differentiation and form bone in vivo

BackgroundInduced pluripotent stem cells (iPSC) are generated by reprogramming somatic cells into embryonic like state (ESC) using defined factors. There is great interest in these cells because of their potential for application in regenerative medicine.ResultsiPSC reprogrammed from murine tail tip fibroblasts were exposed to retinoic acid alone (RA) or in combination with TGF-β1 and 3, basic fibroblast growth factor (bFGF) or bone morphogenetic protein 2 (BMP-2). The resulting cells expressed selected putative mesenchymal stem cells (MSCs) markers; differentiated toward osteoblasts and adipocytic cell lineages in vitro at varying degrees. TGF-beta1 and 3 derived-cells possessed higher potential to give rise to osteoblasts than bFGF or BMP-2 derived-cells while BMP-2 derived cells exhibited a higher potential to differentiate toward adipocytic lineage. TGF-β1 in combination with RA derived-cells seeded onto HA/TCP ceramics and implanted in mice deposited typical bone. Immunofluorescence staining for bone specific proteins in cell seeded scaffolds tissue sections confirmed differentiation of the cells into osteoblasts in vivo.ConclusionsThe results demonstrate that TGF-beta family of proteins could potentially be used to generate murine iPSC derived-cells with potential for osteoblasts differentiation and bone formation in vivo and thus for application in musculoskeletal tissue repair and regeneration.

Abstract 3353: Nodal signaling mediates the aggressive, tumorigenic phenotype in breast cancer cells

Abstract The embryonic morphogen Nodal is a potent inducer of EMT and critical mediator of stem cell pluripotency in the developing embryo, but is not expressed in normal adult tissues. Intriguingly, our lab has recently shown that Nodal, a TGF-beta family member, is in fact secreted by melanoma and breast cancer cells and that Nodal expression positively correlates with tumor grade in human patient samples. Our current studies demonstrate a positive role for Nodal in regulating breast cancer cell renewal and aggressiveness. Specifically, short hairpin (shRNA) knockdown of Nodal expression in human breast cancer cell lines diminishes proliferation, invasion and clonogenicity of these cell lines in vitro and dramatically curtails tumor formation in a mouse xenograft model. Additionally, our data suggest that these effects may be caused, in part, by alterations in the expression of adhesion receptors known to function in tumor engraftment and metastasis. Taken together, our data suggest that Nodal signaling may facilitate the acquisition of self-renewal and migratory capabilities in breast cancer cells and represent a novel therapeutic target for disrupting these processes in breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3353. doi:1538-7445.AM2012-3353

Deleterious interaction between BMP2 and TGF-beta signaling in articular chondrocytes: harmful role for BMP2 in OA?

Purpose: TGF-beta signaling via Smad2/3 is crucial for cartilage maintenance. Smad2/3 maintains a healthy chondrocyte phenotype by inhibiting chondrocyte hypertrophy. We have previously shown that loss of Smad2/3 signaling is a hallmark of both ageing and OA. BMP2, a TGF beta family member, signals via Smad1/5/8 instead of Smad2/3. During OA we found that BMP2 is elevated near lesions. We have investigated whether BMP2, and as a consequence Smad1/5/8 signaling, affects Smad2/3 signaling and what are the biological consequences of this. Methods: Primary bovine chondrocytes were exposed to BMP2 followed by Western blotting for Smad2/3P after 60, 180 and 420 minutes. To study whether the observed effects were reproducible in vivo we injected Ad-BMP2 i.a. into murine knee joints; Ad-luc was used as a control. We isolated knee joints after 3 days for immunohistochemistry on Smad2/3P. To address biological relevance of decreased Smad2/3P, human OA cartilage explants obtained from patients undergoing joint replacement surgery after informed consent were exposed to the Smad2/3P inhibitor SB-505124. After 48 hours RNA was isolated to investigate MMP13 levels as a marker for hypertrophy and OA marker. In addition, we exposed primary bovine chondrocytes to BMP2 and evaluated whether that had an effect on MMP13 levels as well.As we observed that BMP2 reduced Smad2/3P signaling and stimulated MMP13 expression we wanted to investigate whether this effect could be reversed by extra stimulation of the Smad2/3 route. Therefore chondrocytes were exposed to a combination of BMP2 and TGF-beta. Moreover, human OA cartilage explants were exposed to TGF-beta to investigate whether this reduced MMP13 expression. Results: Exposing primary bovine chondrocytes to 15 or 50 ng BMP2 led to a decrease in Smad2/3 phosphorylation on Western Blot already after 60 minutes and that was sustained until at least 420 minutes compared to non-stimulated controls. We confirmed this effect in vivo as over expression of BMP2 in murine knee joints led to decreased Smad2/3P positive cells in both patella and tibia by 55% and 43% respectively. To investigate the functional consequence of decreased Smad2/3 signaling we cultured human OA cartilage explants in the presence of SB-505124. This led to a significant increase in MMP13 expression of 13,6 fold (p<0.02). A similar increase in MMP13 expression was also observed in cultures of primary bovine chondrocytes 48 hours after stimulation with BMP2. Interestingly, the effect of either 15 or 50 ng BMP2 could be prevented by addition of TGF-beta, which prevented the decrease in Smad2/3P levels, but only when a dose of 10 ng/ml TGF-beta was used. A lower dose of 0.5 ng/ml TGF-beta was insufficient to overcome the BMP2-induced Smad2/3P decrease. This indicates that TGF-beta can overcome the BMP2 effect in a dose-dependent manner. Even in human OA cartilage that had spontaneously reduced Smad2/3P levels addition of 10ng/ml TGF-beta could still decrease MMP13 expression by 6.6 fold (p<0.005). Conclusions: Our data confirm that BMP2 down regulates Smad2/3 signaling in articular chondrocytes both in vitro and in vivo. A decreased Smad2/3 signaling in turn results in up regulated levels of MMP13 expression suggesting that BMP2 up regulation, as found adjacent to OA lesions, is deleterious for articular cartilage. Moreover, we show that this negative effect of BMP2 can be overcome by addition of TGF-beta, thereby raising levels of Smad2/3P. Even in human OA cartilage that has naturally high levels of MMP13 and low levels of Smad2/3P, TGF-beta addition can still reduce MMP13 expression levels. Overall our data suggest that BMP2 induces OA-like effects in articular cartilage, down regulating Smad2/3P and up regulating MMP13, which can be overcome by enhanced TGF-beta signaling.

TGFbeta Family Members Are Key Mediators in the Induction of Myofibroblast Phenotype of Human Adipose Tissue Progenitor Cells by Macrophages

ObjectiveThe present study was undertaken to characterize the remodeling phenotype of human adipose tissue (AT) macrophages (ATM) and to analyze their paracrine effects on AT progenitor cells.Research Design and MethodsThe phenotype of ATM, immunoselected from subcutaneous (Sc) AT originating from subjects with wide range of body mass index and from paired biopsies of Sc and omental (Om) AT from obese subjects, was studied by gene expression analysis in the native and activated states. The paracrine effects of ScATM on the phenotype of human ScAT progenitor cells (CD34+CD31−) were investigated.ResultsTwo main ATM phenotypes were distinguished based on gene expression profiles. For ScAT-derived ATM, obesity and adipocyte-derived factors favored a pro-fibrotic/remodeling phenotype whereas the OmAT location and hypoxic culture conditions favored a pro-angiogenic phenotype. Treatment of native human ScAT progenitor cells with ScATM-conditioned media induced the appearance of myofibroblast-like cells as shown by expression of both α-SMA and the transcription factor SNAIL, an effect mimicked by TGFβ1 and activinA. Immunohistochemical analyses showed the presence of double positive α-SMA and CD34 cells in the stroma of human ScAT. Moreover, the mRNA levels of SNAIL and SLUG in ScAT progenitor cells were higher in obese compared with lean subjects.ConclusionsHuman ATM exhibit distinct pro-angiogenic and matrix remodeling/fibrotic phenotypes according to the adiposity and the location of AT, that may be related to AT microenvironment including hypoxia and adipokines. Moreover, human ScAT progenitor cells have been identified as target cells for ScATM-derived TGFβ and as a potential source of fibrosis through their induction of myofibroblast-like cells.

Murine GASP-1 N-Glycosylation is not Essential for its Activity on C2C12 Myogenic Cells but Alters its Secretion

Background/Aims: Growth and differentiation factor-associated serum protein-1 (GASP-1) is a secreted protein known to be capable of binding and inhibiting the activity of several TGF-beta family members, including myostatin. The present study was designed to characterize murine GASP-1 post-translational modifications and to determine their influence on the biological activity of GASP-1. Methods: We describe herein the site-directed mutagenesis of single N-glycosylation sites and combinations of them in 4 mutants of murine GASP-1. Results: In vitro and in vivo analysis revealed that GASP-1 is a glycoprotein containing 2 N-glycans and several mucin-type O-glycans. Treatments by the recombinant murine GASP-1 protein enhance C2C12 proliferation and differentiation by inhibition of the myostatin pathway. The loss of N-glycans leads to a decrease in protein secretion rate but does not affect its ability to activate myogenesis. Conclusion: Analysis of structure-function relationships of murine GASP-1 provides insights into the involvement of the carbohydrate moiety of mGASP-1 on its biological activity.

Abstract 5147: Tumor angiogenesis-promoting activity of a hypoxia-inducible cytokine, growth differentiation factor-15 (GDF-15)

Abstract Angiogenesis, growth of new blood vessels from pre-existing capillary blood vessels, is a marker of cancer and various ischemic and inflammatory diseases. Tumor growth depends on new blood vessel formation because tumor cells require oxygen and nutrients for their survival and proliferation. GDF-15, also known as MIC-1, NAG-1 and PTGF-beta, is a divergent member of the TGF-beta super family. GDF-15, like other TGF-beta family members, has shown janus-faced function in tumor development: pro-apoptotic and anti-tumorigenic vs. tumorigenic activity. Previously, we and others found that hypoxia-condition induced GDF-15 expression in various cancer cell types, leading to increased secretion from the cancer cells. Here, we present the tumor-promoting activity of GDF-15, along with pro-angiogenic activity. In in vivo mouse xenograft model using human melanoma cells, we found that tumors from GDF-15-expressing melanoma cells show higher degree of vessel formation with larger tumor volume than control tumors. Blood vessel inducing activity of GDF-15 was also observed when using human umbilical vein endothelial cells (HUVECs). GDF-15 treatment induced tube formation of HUVECs. Moreover, treatment of GDF-15 enhanced the growth and migration of HUVECs. Blood vessel inducing activity of GDF-15 was further confirmed by CAM and aortic ring sprouting assays, intravital microscopic analysis and matrigel plug assay. Furthermore, GDF-15 transgenic mice showed increased blood vessel development than wild type mice while lymphatic vessel development was no different. Interestingly, although angiogenic activity of GDF-15 was comparable to that of VEGF, its inflammatory activity was less significant than VEGF. Taken together, we show that GDF-15 functions as a potential angiogenic factor for tumor development with seemingly little inflammatory activity. Key words: Cytokine, GDF-15, Tumor, Angiogenesis, Inflammation Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5147. doi:10.1158/1538-7445.AM2011-5147