Transforming growth factor beta: an autocrine regulator of chondrocytes

Transforming growth factor beta (TGF-beta) is a ubiquitous regulator of cellular growth and differentiation. The present study investigated the effects of TGF-beta on chick growth plate chondrocyte proliferation, matrix synthesis, and alkaline phosphatase activity in short term cultures. TGF-beta markedly stimulated DNA synthesis in a dose-dependent manner, while collagen synthesis and cellular and matrix vesicle alkaline phosphatase activity were inhibited. Biologic effects of TGF-beta were correlated with binding to specific receptors, and both high and low affinity receptors were identified. Countercurrent centrifugal elutriation was used to fractionate growth plate chondrocytes to obtain populations of cells in different stages of maturation (effectively from different zones of the growth plate). TGF-beta showed increasing mitogenicity with increasing cellular maturation in the growth plate, with maximal stimulation in the proliferating and early hypertrophic cells. The smallest cells expressed only the high affinity receptor, while with hypertrophy there was increasing expression of the low affinity receptor and a progressive increase in the number of both receptors per cell. Furthermore, the dose-response curves for TGF-beta-stimulated DNA synthesis were not biphasic in the smaller cells, but became progressively more biphasic with cellular hypertrophy and expression of the low affinity receptor. Finally, TGF-beta activity was identified in partially purified chondrocyte conditioned medium by specific bioassay, indicating TGF-beta production by growth plate chondrocytes. The data suggests a potentially important autocrine function for TGF-beta in modulating chondrocyte proliferation and matrix synthesis in endochondral calcification.     

Identification and Analysis of Transforming Growth Factor Beta Receptors on Primary Osteoblast-Enriched Cultures Derived from Adult Human Bone

Primary human osteoblast-enriched (PHO) cultures derived from adult trabecular bone were analyzed to determine the presence or absence of transforming growth factor beta (TGF-β) receptors. Saturation binding studies were performed with ¹²⁵I-TGF-β in the absence or presence of 200-fold excess cold TGF-β. Cross-linking experiments utilizing ¹²⁵-I-TGF-β were performed to identify specific cell surface binding proteins for TGF-β. The saturation binding studies demonstrated saturable binding for TGF-β on PHO cells. TGF-β was cross-linked to cell surface binding proteins of 50 to 110 KDa and a high molecular weight component. Thus, these receptors appear to be similar in affinity, number per cell, and molecular weight to those previously identified with other cell types. The potential biological effects of TGF-β on the growth of PHO cultures were evaluated by both ³H-thymidine incorporation and cell number determination. Growth of PHO cells in the presence of TGF-β resulted in an approximately two-fold stimulation in cell number as compared to control cells while the ³H-thymidine experiments demonstrated a two to four-fold increase in thymidine uptake in the presence of TGF-β. Radiographic emulsion studies revealed that the alkaline phosphatase positive and negative cell populations were responsive to the TGF-β mitogenic stimulation. The cumulative findings of saturable binding, specific cell surface binding proteins, and biological effects suggest that functional TGF-β cell surface receptors are present on primary osteoblast-enriched cultures derived from adult human trabecular bone.     

Modulation of Transforming Growth Factor-β Actions in Rat Osteoblast-like Cells: The Effects of bFGF and EGF

Abstract

The present study examines how the mitogenic and differentiation functions of transforming growth factor-β (TGF-β) are modulated by basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in primary cultures of rat osteoblast-like (ROB) cells. TGF-β, bFGF, and EGF individually stimulated [³H]thymidine incorporation and cell proliferation in a dose range of 0.01-10 ng/ml. When studied in combination, high doses of bFGF and EGF were additive to low doses of TGF-β. The additive effects of bFGF and EGF on mitogenesis diminished with increasing doses of TGF-β. These three factors also decreased alkaline phosphatase activity individually within the same dose range. When cells were treated with the combined factors, only high doses of bFGF and EGF were additive to the TGF-β inhibition. We were unable to detect any change in collagen synthesis with each individual factor or in combined treatments. In addition, TGF-β or bFGF alone or in combination did not affect fibronectin synthesis. Our studies showed that the biological functions of TGF-β can be modulated by bFGF and EGF in ROB cells. The pattern of modulation is varied depending on the specific function examined.     

Engineering endochondral bone: in vitro studies

Chitosan scaffolds have been shown to possess biological and mechanical properties suitable for tissue engineering and clinical applications. In the present work, chitosan sponges were evaluated regarding their ability to support cartilage cell proliferation and maturation, which are the first steps in endochondral bone formation. Chitosan sponges were seeded with chondrocytes isolated from chicken embryo sterna. Chondrocyte/chitosan constructs were cultured for 20 days, and treated with retinoic acid (RA) to induce chondrocyte maturation and matrix synthesis. At different time points, samples were collected for microscopic, histological, biochemical, and mechanical analyses. Results show chondrocyte attachment, proliferation, and abundant matrix synthesis, completely obliterating the pores of the sponges. RA treatment caused chondrocyte hypertrophy, characterized by the presence of type X collagen in the extracellular matrix and increased alkaline phosphatase activity. In addition, hypertrophy markedly changed the mechanical properties of the chondrocyte/chitosan constructs. In conclusion, we have developed chitosan sponges with adequate pore structure and mechanical properties to serve as a support for hypertrophic chondrocytes. In parallel studies, we have evaluated the ability of this mature cartilage scaffold to induce endochondral ossification.     

Longitudinal bone growth is impaired by direct involvement of caffeine with chondrocyte differentiation in the growth plate

We showed previously that caffeine adversely affects longitudinal bone growth and disrupts the histomorphometry of the growth plate during the pubertal growth spurt. However, little attention has been paid to the direct effects of caffeine on chondrocytes. Here, we investigated the direct effects of caffeine on chondrocytes of the growth plate in vivo and in vitro using a rapidly growing young rat model, and determined whether they were related to the adenosine receptor signaling pathway. A total of 15 male rats (21 days old) were divided randomly into three groups: a control group and two groups fed caffeine via gavage with 120 and 180 mg kg⁻¹ day⁻¹ for 4 weeks. After sacrifice, the tibia processed for the analysis of the long bone growth and proliferation of chondrocytes using tetracycline and BrdU incorporation, respectively. Caffeine‐fed animals showed decreases in matrix mineralization and proliferation rate of growth plate chondrocytes compared with the control. To evaluate whether caffeine directly affects chondrocyte proliferation and chondrogenic differentiation, primary rat chondrocytes were isolated from the growth plates and cultured in either the presence or absence of caffeine at concentrations of 0.1–1 mm , followed by determination of the cellular proliferation or expression profiles of cellular differentiation markers. Caffeine caused significant decreases in extracellular matrix production, mineralization, and alkaline phosphatase activity, accompanied with decreases in gene expression of the cartilage‐specific matrix proteins such as aggrecan, type II collagen and type X. Our results clearly demonstrate that caffeine is capable of interfering with cartilage induction by directly inhibiting the synthetic activity and orderly expression of marker genes relevant to chondrocyte maturation. In addition, we found that the adenosine type 1 receptor signaling pathway may be partly involved in the detrimental effects of caffeine on chondrogenic differentiation, specifically matrix production and mineralization, as evidenced by attenuation of the inhibitory effects of caffeine by blockade of this receptor. Thus, our study provides novel information on the integration of caffeine and adenosine receptor signaling during chondrocyte maturation, extending our understanding of the effect of caffeine at a cellular level on chondrocytes of the growth plate.     

The Use of Growth Factors in the Proliferation of Avian Articular Chondrocytes in a Serum-Free Culture System

The purpose of this research was to develop a serum-free culture system for the proliferation of articular chondrocytes. Various growth factors and hormones were tested for their ability to stimulate avian articular chondrocyte proliferation in a defined, serum-free media. Multiple members of the fibroblast growth factor (FGF) family (FGFs: 2, 4, and 9), insulin-like growth factor-1 (IGF-1) and transforming growth factor β (TGF-β) significantly stimulated ³H-thymidine uptake by chondrocytes grown in an adherent serum-free, culture system. Double or triple combinations of these mitogenic growth factors further stimulated cell proliferation to levels that were equivalent to, or surpassed those of cells grown in serum. Although proliferation was maximally stimulated, chondrocytes grown in the presence of FGF-2, IGF-1, and TGF-β, began to exhibit changes in morphology and collagen II expression declined. This culture system could be used to rapidly expand a population of articular chondrocytes prior to transferring these cells to a non-adherent culture system, which could then stabilize the chondrocyte phenotype and maximize matrix synthesis and integrity.     

转化生长因子-β在心脏重构中作用的研究进展

由于对心肌、心肌间质细胞及免疫细胞的影响,转化生长因子-β(TGF-β)在心脏重构和心肌纤维化过程中发挥关键作用.小鼠心肌TGF-β过表达与心肌纤维化和心脏扩大相关.内源性TGF-β在心脏纤维化和肥厚重构过程中具有关键作用,且对超负荷心脏基质新陈代谢具有调整作用.在梗塞心肌中,TGF-β可以灭活炎症巨噬细胞,同时通过信...     

Differential expression of biological effects in maturationally distinct subpopulations of growth plate chondrocytes

Countercurrent centrifugal elutriation is an accurate and reproducible technique which can separate cells on the basis of size. This technique was utilized for the separation of isolated chick growth plate chondrocytes. Mean cellular volume, alkaline phosphatase activity, and Type X collagen synthesis progressively increased in each of seven successive separated fractions. The chondrocytes from the different fractions expressed a differential response to the growth regulator, transforming growth factor beta (TGF beta). TGF beta receptor binding to these cell fractions suggests that the different biological effects may be due to differences in the number of receptors expressed per cell as well as to differences in the proportion of high and low affinity TGF beta receptors present.     

Prodomains regulate the synthesis,extracellular localisation and activity of TGF-β superfamily ligands

All transforming growth factor-β (TGF-β) ligands are synthesised as precursor molecules consisting of a signal peptide, an N-terminal prodomain and a C-terminal mature domain. During synthesis, prodomains interact non-covalently with mature domains, maintaining the molecules in a conformation competent for dimerisation. Dimeric precursors are cleaved by proprotein convertases, and TGF-β ligands are secreted from the cell non-covalently associated with their prodomains. Extracellularly, prodomains localise TGF-β ligands within the vicinity of their target cells via interactions with extracellular matrix proteins, including fibrillin and perlecan. For some family members (TGF-β1, TGF-β2, TGF-β3, myostatin, GDF-11 and BMP-10), prodomains bind with high enough affinity to suppress biological activity. The subsequent mechanism of activation of these latent TGF-β ligands varies according to cell type and context, but all activating mechanisms directly target prodomains. Thus, prodomains control many aspects of TGF-β superfamily biology, and alterations in prodomain function are often associated with disease.     

Matrix vesicles as a marker of endochondral ossification

Endochondral ossification in bone development and repair, and in induced bone formation in mesenchymal tissues, involves recruitment of mesenchymal cells, their differentiation into chondrocytes, and calcification of the cartilagenous matrix. Stimulation of proteoglycan synthesis is used as a biochemical marker of chondrogenesis, however it does not distinguish among chondrogenic phenotypes. Chondrocytes derived from the resting zone and adjacent growth zone cartilage of the costochondral junction of young rats, produce matrix vesicles in culture which are enriched in alkaline phosphatase specific activity with respect to the plasma membrane. Matrix vesicles isolated from cultures of neonatal rat muscle mesenchymal cells are not enriched in this enzyme activity. Alkaline phosphatase in matrix vesicles produced by growth zone chondrocytes is stimulated by 1,25(OH)2D3; enzyme in matrix vesicles produced by resting zone chondrocytes is stimulated by 24,25(OH)2D3; enzyme in matrix vesicles isolated from mesenchymal cell cultures is responsive to neither metabolite. Matrix vesicle phospholipase A2 is stimulated by 1,25(OH)2D3 in growth zone chondrocytes cultures; inhibited by 24,25(OH)2D3 in resting zone chondrocyte cultures; and is unaffected by either metabolite in mesenchymal cell cultures. These observations suggest that matrix vesicle production, as defined by alkaline phosphatase enrichment, and responsiveness of matrix vesicle enzymes to vitamin D metabolites, can be used as markers of phenotypic maturation during chondrogenesis in vivo and in vitro.     

Cytochemical localization of tartrate-resistant acid phosphatase,alkaline phosphatase,and nonspecific esterase in perivascular cells of cartilage canals in the developing mouse epiphysis

Cytochemical localization of tartrate-resistant acid phosphatase (TRAP), tartratc-sensitive acid phosphatases (TSAP), alkaline phosphatase, and nonspecific esterase was used to characterize perivascular cells within cartilage canals. In the distal femoral epiphyses of 5- to 7-day-old mice, three stages of canal development can be distinguished, and at each developmental stage different perivascular cells were present with morphological characteristics of degradative cells. Vacuolated cells resembling macrophages, fibroblastic cells, and chondroclasts were present adjacent to the matrix in superficial, intermediate, and deep canals, respectively. In order to characterize these perivascular cells cytochemically, nonspecific esterase and TSAP staining was used to identify macrophages, alkaline phosphatase staining was used to identify fibroblastic cells, and TRAP staining was used to identify chondroclasts. There were no cells present in the canals at any developmental stage that were positive for TSAP or strongly positive for nonspecific esterase, placing doubt on the identity of the vacuolated cells as macrophages. Alkaline phosohatase-positive perivascular cells were present in the intermediate and deep canals adjacant to matrix containing alkaline phosphatase-positive chondrocytes. These alkaline phosphatase-positive cells were found in the same location within canals as the fibroblastic cells. Tartrate-resislant acid phosphatase was localized in chondroclasts at the tips of deep canals but was not confined exclusively to chondroclasts. Except for the very early stage of canal development prior to chondrocyte hypertrophy, TRAP-positive cells were present at the tips of superficial and intermediate canals as well as at the tips of the deep canals. Additionally, the presence of TRAP in chondrocytes with in the growth plate, in chondrocytes within the epiphyseal cartilage near some canals, and in perichondrial cells suggests that TRAP is associated with matrix degradation in the cartilage.     

Monokines Produced by Macrophages Stimulate the Growth of Osteoblasts

We have previously reported that the J774A.1 macrophage-like tumor cell line produces two potent monokines which stimulate the growth of osteoblasts and chondrocytes. These growth factors, which have an affinity for heparin-agarose, have been termed HEP I (a 30 Kd PDGF-like molecule) and HEP II (an approximately 20 Kd molecule), respectively, based on their elution profile. Unlike HEP I, HEP II does not stimulate the growth of fibroblasts. Extensive biological and chromatographic studies disclosed that HEP II appears to be a unique bone cell mitogen unlike any known growth factor, including the FGFs, IL-Is, and TNFs, EGF, IGF-I and -II, TGF-β, β2 microglobulin, G-CSF, CSF-I and GM-CSF. To characterize more fully the effects of the macrophage-derived monokines on osteoblast growth and function, clones were derived from calvaria explant cultures. Two clones, SDFRC-2.05 and SDFRC-3, were developed and found to exhibit osteoblastic characteristics, including high levels of alkaline phosphatase, synthesis of type I but not type III collagen, and an increased intracellular cAMP production in response to PTH. The SDFRC-3 cells exhibited a polygonal morphology like that of the explant-derived cells while SDFRC-2.05 cells exhibited a more fibroblastic morphology. When tested on the explant cultures and clones, HEP I and HEP II were found to stimulate DNA synthesis and increase protein per culture, but decreased alkaline phosphatase activity. Clone SDFRC-3 was found to be more responsive to HEP II than clone SDFRC-2.05. Both monokines were found to be more potent mitogens for bone cells than TGF-β, HEP II, but not HEP I or TGF-β, induced a transformation of bone cells from a polygonal to a fibroblastic morphology, suggesting the induction of migration prior to proliferation. Thus, macrophages may be responsible not only for bone repair but also for ensuring the linkage of bone formation to resorption during physiological remodeling.     

Interplay between local versus soluble transforming growth factor-beta and fibrin scaffolds: role of cells and impact on human mesenchymal stem cell chondrogenesis

Structural extracellular matrix molecules gain increasing attention as scaffolds for cartilage tissue engineering owing to their natural role as a growth factor repository. We recently observed that a collagen-type I/III (Col-I/III) matrix, human recombinant transforming growth factor-beta (TGF-β) protein, and fibrin hydrogel (FG) combined to a biphasic construct provided sufficient long-term TGF-β support to drive in vitro chondrogenesis of human mesenchymal stem cells (hMSC). Here we ask whether FG and Col-I/III can both retain TGF-β, describe the influence of cell seeding on TGF-β release, and compare the molecular path of hMSC chondrogenic differentiation under soluble versus local TGF-β supply. Release of growth factor from scaffolds augmented with increasing amounts of TGF-β was analyzed over 7 days and chondrogenesis was assessed over 42 days. Low TGF-β release rates from Col-I/III as opposed to higher release from FG indicated that both molecules retained TGF-β, with Col-I/III being the superior storage component. Cell seeding enhanced TGF-β retention in FG by about threefold and almost stopped release beyond 24?h. TGF-β remained bioactive and supported MSC chondrogenesis without impairing the amount of proteoglycan and collagen-type II deposition per cell and per construct compared to standard scaffold-free MSC pellets supplied with soluble TGF-β. Local TGF-β, however, mediated lower cell content, less collagen-type X relative to collagen-type II deposition and no matrix metalloproteinase-13 up-regulation. In conclusion, cells quickly halted release of local TGF-β from FG, turning FG and Col-I/III into attractive TGF-β repositories capable to drive full hMSC chondrogenesis, but via a modulated differentiation pathway. Since only part of the changes was reproduced by transient soluble TGF-β supply, release kinetics alone could not explain the molecular differences, suggesting that local TGF-β acts distinct from its soluble counterpart.     

Biphasic calcium phosphate: a scaffold for growth plate chondrocyte maturation

While skeletal development can occur by either intramembranous or endochondral bone formation, all current tissue engineering approaches for bone repair and regeneration try to mimic intramembranous ossification. In this study, we propose to create an in vitro cartilage template as the transient model for in vivo endochondral bone formation. The goals of this study are to (1) establish a method of growing chondrocytes in a well-characterized macroporous biphasic calcium phosphate (MBCP) scaffold and (2) induce maturation of chondrocytes grown in the MBCP scaffold. Chondrocytes isolated from chick embryonic tibia were grown on MBCP particles and treated with retinoic acid to induce chondrocyte maturation and extracellular matrix deposition. Chondrocytes were observed to attach and proliferate on the MBCP scaffold. The thickness of the chondrocyte and extracellular matrix layer increased in the presence of the retinoid. Alkaline phosphatase activity and expression, proteoglycans synthesis, cbfa1 and type I collagen mRNA levels also increased in the presence of retinoic acid. These results demonstrated for the first time the proliferation, maturation of chondrocytes, and matrix deposition on MBCP, suggesting the potential for such scaffold in tissue engineering via the endochondral bone formation mechanism.     

RAP-PCR fingerprinting reveals time-dependent expression of matrix-related molecules following stem-cell based TGFβ1-induced chondrocyte development

Different approaches of engineering cartilage to treat defects in the articulating surfaces of the joints have been designed, which mainly use mesenchymal stem cells or autologous chondrocytes for in situ transplantation. However, these cells are poorly characterized with respect to viability, degree of differentiation and morphology or production of extracellular matrix. At present, one of the key approaches to generate chondrocytes is the stimulation of stem cells with transforming growth factor (TGF) β1. To characterize the molecular alterations occurring during the cellular transformation induced by TGF-β1 exposure, the differentiation process of bone marrow-derived stem cells into chondrocytes was investigated using an in vitro chondrogenesis model and the RNA arbitrarily primed PCR (RAP-PCR) fingerprinting technique. Distinct genes were found to be differentially regulated during chondrocyte development beginning on day 1: collagen type I, non-muscle myosin MYH9, followed by manganese superoxide dismutase and sodium-potassium ATPase on day 7. The results suggest that using RAP-PCR for differential display fingerprinting is a useful tool to investigate the differentiation process of bone marrow-derived stem cells following TGF-β1-stimulation.     

Studies on the pathogenesis of avian rickets. I. Changes in epiphyseal and metaphyseal vessels in hypocalcemic and hypophosphatemic rickets.

Growth plate morphometry and measurements of serum chemistry were correlated to clarify the pathogenesis of hypocalcemic and hypophosphatemic avian rickets. Accumulation of proliferating and maturing cartilage in hypocalcemic chicks is accompanied by increased length and increased variation in length of perforating epiphyseal vessels, decreased number and abnormal arrangement of marrow spaces, an increased proportion of cells to blood vessels in marrow spaces, and a change in the distribution but not the total number of DNA-synthesizing chondrocytes per unit width of growth plate. Accumulation of hypertrophic cartilage in hypophosphatemic rickets is accompanied by no change in length, distribution, or number of perforating epiphyseal vessels, elongation but no change in number or arrangement of marrow spaces, an increase in the relative proportion of blood vessels to cells in the marrow spaces, and no change in distribution but a decrease in total number of DNA-synthesizing chondrocytes per unit width of growth plate. Both types of rickets have decreased amounts of calcified cartilage. These results provide further evidence that hypocalcemia and hypophosphatemia cause morphologically distinct types of rickets in birds. The data indicate that the thickness of the proliferating and maturing region and hence the distance of the hypertrophic zones from the epiphysis are anatomically and temporally related to length of perforating epiphyseal vessels and serum calcium levels. They indicate that in hypocalcemic rickets accumulation of proliferating and maturing cartilage is unlikely to be the result of increased chondrocyte replication and that the relative rates of chondrocyte hypertrophy and resorption of hypertrophic cartilage by marrow are equal. They support the concept that delayed chondrocyte hypertrophy is the major cause of growth plate thickening in hypocalcemic rickets. Data presented in this study, when considered together with data from the literature on hypophosphatemic rickets, support the long-held concept that growth plate thickening in this disease is caused primarily by a decreased rate of resorption of hypertrophic cartilage by marrow relative to the rates of chondrocyte proliferation, maturation, and hypertrophy. The data further support the concepts that growth of cartilage into marrow is a biphasic process including longitudinal growth effected mainly by blood vessels, and resorption of the lateral walls of marrow spaces effected mainly by marrow cells, and that it is the latter phase that is defective in hypophosphatemia.     

Prodomains regulate the synthesis, extracellular localisation and activity of TGF-β superfamily ligands

All transforming growth factor-β (TGF-β) ligands are synthesised as precursor molecules consisting of a signal peptide, an N-terminal prodomain and a C-terminal mature domain. During synthesis, prodomains interact non-covalently with mature domains, maintaining the molecules in a conformation competent for dimerisation. Dimeric precursors are cleaved by proprotein convertases, and TGF-β ligands are secreted from the cell non-covalently associated with their prodomains. Extracellularly, prodomains localise TGF-β ligands within the vicinity of their target cells via interactions with extracellular matrix proteins, including fibrillin and perlecan. For some family members (TGF-β1, TGF-β2, TGF-β3, myostatin, GDF-11 and BMP-10), prodomains bind with high enough affinity to suppress biological activity. The subsequent mechanism of activation of these latent TGF-β ligands varies according to cell type and context, but all activating mechanisms directly target prodomains. Thus, prodomains control many aspects of TGF-β superfamily biology, and alterations in prodomain function are often associated with disease.     

Transforming growth factor-beta: A target for cancer therapy

Transforming growth factor-beta (TGF-β) is a pleiotropic growth factor that regulates cell growth and differentiation, apoptosis, cell motility, extracellular matrix production, angiogenesis, and cellular immune responses. TGF-β demonstrates paradoxical action whereby it can function to suppress early tumorigenesis; however, it can also facilitate malignant transformation and stimulate tumor growth by manipulating a more hospitable environment for tumor invasion and the development of metastases. Given the integral role of TGF-β in transformation and cancer progression, various components of the TGF-β signaling pathway offer potentially attractive therapeutic targets for cancer treatment. This review focuses on the role of TGF-β in cancer and discusses both small and large molecule drugs currently in development that target TGF-β, its receptor and important down stream steps along its signaling pathway.