ABSTRACT
The insulin-like growth factor-1 system, including its critical mediator insulin receptor substrate-1 (IRS-1), is involved in regulating osteosarcoma (OS) cell proliferation or differentiation. The aim of this study is to define the role of IRS-1 in OS cells by assessing the contribution of IRS-1 in the differentiation of human and murine OS cell lines and mouse mesenchymal stem cells (MSCs) and found that the basal level of IRS-1 is important for the initiation of differentiation. Both down-regulation and over-expression of IRS-1 inhibited osteoblastic differentiation. In vivo studies showed that OS cells over-expressing IRS-1 have increased metastatic potential and tumor growth. The proteasome inhibitor MG-132 led to an increase in IRS-1 protein level that inhibited osteoblastic differentiation, suggesting a role for proteasomal regulation in maintaining the appropriate expression level of IRS-1. Thus, precise regulation of IRS-1 expression level is critical for determining the differentiating capacity of MSCs and OS cells, and that derangement of IRS-1 levels can be a critical step in OS transformation.
Subject(s)
Insulin Receptor Substrate Proteins/biosynthesis , Insulin-Like Growth Factor I/metabolism , Mesenchymal Stem Cells/metabolism , Osteoblasts/metabolism , Osteosarcoma/pathology , Animals , Cell Differentiation/genetics , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Cysteine Proteinase Inhibitors/pharmacology , Gene Expression Regulation, Developmental/genetics , Gene Expression Regulation, Neoplastic/genetics , Humans , Insulin Receptor Substrate Proteins/genetics , Insulin Receptor Substrate Proteins/metabolism , Leupeptins/pharmacology , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, Nude , Osteocalcin/biosynthesis , Phosphorylation , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , RNA Interference , RNA, Small Interfering , Signal Transduction/genetics , Sp7 Transcription Factor , Transcription Factors/biosynthesisABSTRACT
TGF-ß type II receptor (Tgfbr2) signaling plays an essential role in joint-element development. The Tgfbr2(PRX-1KO) mouse, in which the Tgfbr2 is conditionally inactivated in developing limbs, lacks interphalangeal joints and tendons. In this study, we used the Tgfbr2-ß-Gal-GFP-BAC mouse as a LacZ/green fluorescent protein (GFP)-based read-out to determine: the spatial and temporally regulated expression pattern of Tgfbr2-expressing cells within joint elements; their expression profile; and their slow-cycling labeling with bromodeoxyuridine (BrdU). Tgfbr2-ß-Gal activity was first detected at embryonic day (E) 13.5 within the interphalangeal joint interzone. By E16.5, and throughout adulthood, Tgfbr2-expressing cells clustered in a contiguous niche that comprises the groove of Ranvier and the synovio-entheseal complex including part of the perichondrium, the synovium, the articular cartilage superficial layer, and the tendon's entheses. Tgfbr2-expressing cells were found in the synovio-entheseal complex niche with similar temporal pattern in the knee, where they were also detected in meniscal surface, ligaments, and the synovial lining of the infrapatellar fat pad. Tgfbr2-ß-Gal-positive cells were positive for phospho-Smad2, signifying that the Tgfbr2 reporter was accurate. Developmental-stage studies showed that Tgfbr2 expression was in synchrony with expression of joint-morphogenic genes such as Noggin, GDF5, Notch1, and Jagged1. Prenatal and postnatal BrdU-incorporation studies showed that within this synovio-entheseal-articular-cartilage niche most of the Tgfbr2-expressing cells labeled as slow-proliferating cells, namely, stem/progenitor cells. Tgfbr2-positive cells, isolated from embryonic limb mesenchyme, expressed joint progenitor markers in a time- and TGF-ß-dependent manner. Our studies provide evidence that joint Tgfbr2-expressing cells have anatomical, ontogenic, slow-cycling trait and in-vivo and ex-vivo expression profiles of progenitor joint cells.
Subject(s)
Foot Joints/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptors, Transforming Growth Factor beta/metabolism , Animals , Antigens, Differentiation/metabolism , Cartilage, Articular/metabolism , Cell Proliferation , Cells, Cultured , Female , Foot Joints/cytology , Forelimb/cytology , Forelimb/metabolism , Gene Expression , Gene Expression Regulation, Developmental , Male , Mice , Mice, Transgenic , Protein Serine-Threonine Kinases/genetics , Receptor, Transforming Growth Factor-beta Type II , Receptors, Transforming Growth Factor beta/genetics , Stem Cell Niche , Stem Cells/metabolism , Synovial Membrane/metabolismABSTRACT
The obesity hormone leptin has been implicated in breast cancer development. Breast cancer cells express the leptin receptor and are able to synthesize leptin in response to obesity-related stimuli. Furthermore, leptin is a positive regulator of vascular endothelial growth factor (VEGF) and high levels of both proteins are associated with worse prognosis in breast cancer patients. Peroxisome proliferator-activated receptor γ (PPARγ) ligands are therapeutic agents used in patient with Type 2 diabetes and obesity which have recently been studied for their potential anti-tumor effect. Here, we studied if these compounds, ciglitazone and GW1929, can affect the expression of leptin and VEGF in breast cancer cells. In MDA-MB-231 and MCF-7 breast cancer cells, treatment with submolar concentrations of ciglitazone and GW1929 elevated the expression of leptin and VEGF mRNA and protein, and increased cell viability and migration. These effects coincided with increased recruitment of PPARγ to the proximal leptin promoter and decreased association of a transcriptional factor Sp1 with this DNA region.
Subject(s)
Benzophenones/pharmacology , Breast Neoplasms/metabolism , Leptin/metabolism , PPAR gamma/agonists , Thiazolidinediones/pharmacology , Tyrosine/analogs & derivatives , Vascular Endothelial Growth Factor A/metabolism , Benzophenones/toxicity , Binding Sites , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Movement/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Female , Gene Expression Regulation, Neoplastic , Humans , Leptin/genetics , Ligands , MCF-7 Cells , PPAR gamma/metabolism , Promoter Regions, Genetic , RNA, Messenger/metabolism , Sp1 Transcription Factor/metabolism , Thiazolidinediones/toxicity , Tyrosine/pharmacology , Tyrosine/toxicity , Vascular Endothelial Growth Factor A/geneticsABSTRACT
Despite its clinical significance, the mechanisms of joint morphogenesis are elusive. By combining laser-capture microdissection for RNA sampling with microarrays, we show that the setting in which joint-forming interzone cells develop is distinct from adjacent growth plate chondrocytes and is characterized by downregulation of chemokines, such as monocyte-chemoattractant protein-5 (MCP-5). Using in vivo, ex vivo, and in vitro approaches, we show that low levels of interzone-MCP-5 are essential for joint formation and contribute to proper growth plate organization. Mice lacking the TGF-ß-type-II-receptor (TßRII) in their limbs (Tgfbr2(Prx1KO)), which lack joint development and fail chondrocyte hypertrophy, show upregulation of interzone-MCP-5. In vivo and ex vivo blockade of the sole MCP-5 receptor, CCR2, led to the rescue of joint formation and growth plate maturation in Tgfbr2(Prx1KO) but an acceleration of growth plate mineralization in control mice. Our study characterized the TßRII/MCP-5 axis as an essential crossroad for joint development and endochondral growth.
Subject(s)
Growth Plate/embryology , Joints/embryology , Monocyte Chemoattractant Proteins/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Receptors, Transforming Growth Factor beta/genetics , Receptors, Transforming Growth Factor beta/metabolism , Animals , Cartilage, Articular/cytology , Cartilage, Articular/embryology , Chondrocytes/physiology , Female , Gene Expression Regulation, Developmental/physiology , Growth Plate/cytology , Joints/cytology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Monocyte Chemoattractant Proteins/genetics , Pregnancy , Receptor, Transforming Growth Factor-beta Type II , Signal Transduction/physiologyABSTRACT
In this study, we examined the effectiveness of systemic subcutaneous delivery of recombinant Insulin-like growth factor (IGF)-I concurrently with primary cultured bone marrow-derived mesenchymal stem cell (MSC) transplant on fracture repair. We found that the fracture callus volume increased in mice with a stabilized tibia fracture that received IGF-I+MSC when compared with that in either untreated or MSC alone treated mice. In evaluating the callus tissue components, we found that the soft and new bone tissue volumes were significantly increased in IGF-I+MSC recipients. Histological and in-situ hybridization analyses confirmed a characteristic increase of newly forming bone in IGF-I+MSC recipients and that healing progressed mostly through endochondral ossification. The increase in soft and new bone tissue volumes correlated with increased force and toughness as determined by biomechanical testing. In conclusion, MSC transplant concurrent with systemic delivery of IGF-I improves fracture repair suggesting that IGF-I+MSC could be a novel therapeutic approach in patients who have inadequate fracture repair.
