Shuiliang Shi

Growth Factor Regulation of Growth Factors in Articular Chondrocytes

Several lines of evidence indicate that polypeptide growth factors are important in articular cartilage homeostasis and repair. It is not yet clear how these growth factors are regulated. We tested the hypothesis that the growth factors responsible for regulating cartilage are themselves regulated by growth factors. We delivered insulin-like growth factor I (IGF-I), fibroblast growth factor-2 (FGF-2), and/or transforming growth factor-β1 (TGF-β1) to adult bovine articular chondrocytes in primary culture and measured the resulting changes in IGF-I, FGF-2, and TGF-β1 gene expression and protein production. These growth factors differentially regulated their own and each others gene expression and protein production. In concert, they regulated each other in an interactive fashion. Their interactions ranged from inhibitory to synergistic. The time course of the regulatory effects differed among the individual growth factors and combinations. Growth factor-induced changes in growth factor protein production by articular chondrocytes generally corresponded to the changes in gene expression patterns. These studies suggest that interactions among IGF-I, FGF-2, and TGF-β1 substantially modulate their regulatory functions. The results may help guide the application of growth factors to articular cartilage repair.

Growth factor transgenes interactively regulate articular chondrocytes

Adult articular chondrocytes lack an effective repair response to correct damage from injury or osteoarthritis. Polypeptide growth factors that stimulate articular chondrocyte proliferation and cartilage matrix synthesis may augment this response. Gene transfer is a promising approach to delivering such factors. Multiple growth factor genes regulate these cell functions, but multiple growth factor gene transfer remains unexplored. We tested the hypothesis that multiple growth factor gene transfer selectively modulates articular chondrocyte proliferation and matrix synthesis. We tested the hypothesis by delivering combinations of the transgenes encoding insulin‐like growth factor I (IGF‐I), fibroblast growth factor‐2 (FGF‐2), transforming growth factor beta1 (TGF‐β1), bone morphogenetic protein‐2 (BMP‐2), and bone morphogenetic protien‐7 (BMP‐7) to articular chondrocytes and measured changes in the production of DNA, glycosaminoglycan, and collagen. The transgenes differentially regulated all these chondrocyte activities. In concert, the transgenes interacted to generate widely divergent responses from the cells. These interactions ranged from inhibitory to synergistic. The transgene pair encoding IGF‐I and FGF‐2 maximized cell proliferation. The three‐transgene group encoding IGF‐I, BMP‐2, and BMP‐7 maximized matrix production and also optimized the balance between cell proliferation and matrix production. These data demonstrate an approach to articular chondrocyte regulation that may be tailored to stimulate specific cell functions, and suggest that certain growth factor gene combinations have potential value for cell‐based articular cartilage repair. J. Cell. Biochem. 114: 908–919, 2013.

Regulation of articular chondrocyte aggrecan and collagen gene expression by multiple growth factor gene transfer

Gene transfer is a promising approach to the delivery of chondrotrophic growth factors to promote cartilage repair. It is unlikely that a single growth factor transgene will optimally regulate these cells. The aim of this study was to identify those growth factor transgene combinations that optimally regulate aggrecan, collagen type II and collagen type I gene expression by articular chondrocytes. We delivered combinations of the transgenes encoding fibroblast growth factor‐2, insulin‐like growth factor I, transforming growth factor beta1, bone morphogenetic protein‐2, and/or bone morphogenetic protein‐7 and assessed chondrocyte responses by measuring changes in the expression of aggrecan, type II collagen and type I collagen genes. These growth factor transgenes differentially regulated the magnitude and time course of all three‐matrix protein genes. In concert, the transgenes regulated matrix gene expression in an interactive fashion that ranged from synergistic to inhibitory. Maximum stimulation of aggrecan (16‐fold) and type II collagen (35‐fold) expression was with the combination of IGF‐I, BMP‐2, and BMP‐7 transgenes. The results indicate that the optimal choice of growth factor genes for cell‐based cartilage repair cannot be predicted from observations of individual transgenes. Rather, such gene therapy will require an empirically based selection of growth factor gene combinations. 2011 Orthopaedic Research Society. This article is a US Government work and as such is in the public domain in the United States of America. Published by Wiley Periodicals, Inc. J Orthop Res 30:1026–1031, 2012

Role of Sox9 in growth factor regulation of articular chondrocytes

Chondrogenic polypeptide growth factors influence articular chondrocyte functions that are required for articular cartilage repair. Sox9 is a transcription factor that regulates chondrogenesis, but its role in the growth factor regulation of chondrocyte proliferation and matrix synthesis is poorly understood. We tested the hypotheses that selected chondrogenic growth factors regulate sox9 gene expression and protein production by adult articular chondrocytes and that sox9 modulates the actions of these growth factors. To test these hypotheses, we delivered insulin‐like growth factor‐I (IGF‐I), fibroblast growth factor‐2 (FGF‐2), bone morphogenetic protein‐2 (BMP‐2) and/or bone morphogenetic protein‐7 (BMP‐7), or their respective transgenes to adult bovine articular chondrocytes, and measured changes in sox9 gene expression and protein production. We then knocked down sox9 gene expression with sox9 siRNA, and measured changes in the expression of the genes encoding aggrecan and types I and II collagen, and in the production of glycosaminoglycan, collagen and DNA. We found that FGF‐2 or the combination of IGF‐I, BMP‐2, and BMP‐7 increased sox9 gene expression and protein production and that sox9 knockdown modulated growth factor actions in a complex fashion that differed both with growth factors and with chondrocyte function. The data suggest that sox9 mediates the stimulation of matrix production by the combined growth factors and the stimulation of chondrocyte proliferation by FGF‐2. The mitogenic effect of the combined growth factors and the catabolic effect of FGF‐2 appear to involve sox9‐independent mechanisms. Control of these molecular mechanisms may contribute to the treatment of cartilage damage. J. Cell. Biochem. 116: 1391–1400, 2015.

Effect of transfection strategy on growth factor overexpression by articular chondrocytes

Articular cartilage damage remains an unsolved problem in orthopaedics. Insulin‐like growth factor I (IGF‐I) and fibroblast growth factor‐2 (FGF‐2) are anabolic and mitogenic for articular chondrocytes, and are candidates for the application of gene therapy to articular cartilage repair. We tested the hypothesis that the production of IGF‐I and FGF‐2 can be augmented by modulating vector designs and delivery methods used for gene transfer to articular chondrocytes. We developed a novel adeno‐associated virus (AAV)‐based plasmid (pAAV) to overexpress IGF‐I and FGF‐2 cDNAs in adult bovine articular chondrocytes. We found that the pAAV‐based vectors generated significantly more growth factor than pcDNA vectors carrying the same cDNAs. Chondrocytes cotransfected with both IGF‐I and FGF‐2 cDNAs in two separate pAAV plasmids produced significantly more IGF‐I and FGF‐2 than cells transfected by a single pAAV plasmid carrying both cDNAs in a dicistronic cassette. These data indicate that pAAV vectors are more effective than pcDNA vectors for transfer of IGF‐I and FGF‐2 genes to articular chondrocytes. They further suggest that cotransfection may be an effective strategy for multiple gene transfer to these cells. These findings may be important in applying growth factor gene transfer to cell‐based articular cartilage gene therapy.

Endogenous versus Exogenous Growth Factor Regulation of Articular Chondrocytes

Anabolic growth factors that regulate the function of articular chondrocytes are candidates for articular cartilage repair. Such factors may be delivered by pharmacotherapy in the form of exogenous proteins, or by gene therapy as endogenous proteins. It is unknown whether delivery method influences growth factor effectiveness in regulating articular chondrocyte reparative functions. We treated adult bovine articular chondrocytes with exogenous recombinant insulin‐like growth factor‐I (IGF‐I) and transforming growth factor‐beta1 (TGF‐β1), or with the genes encoding these growth factors for endogenous production. Treatment effects were measured as change in chondrocyte DNA content, glycosaminoglycan production, and aggrecan gene expression. We found that IGF‐I stimulated chondrocyte biosynthesis similarly when delivered by either exogenous or endogenous means. In contrast, exogenous TGF‐β1 stimulated these reparative functions, while endogenous TGF‐β1 had little effect. Endogenous TGF‐β1 became more bioactive following activation of the transgene protein product. These data indicate that effective mechanisms of growth factor delivery for articular cartilage repair may differ for different growth factors. In the case of IGF‐I, gene therapy or protein therapy appear to be viable options. In contrast, TGF‐β1 gene therapy may be constrained by a limited ability of chondrocytes to convert latent complexes to an active form. Published 2013 by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 32:54–60, 2014.

Growth factor regulation of growth factor production by multiple gene transfer to chondrocytes.

Of the many classes of molecules regulated by growth factors, growth factors themselves are not well investigated. We tested the hypothesis that combinations of endogenous growth factors interactively regulate the production of other growth factors. Growth factors have therapeutic potential for articular cartilage repair, and gene transfer is a promising approach to growth factor delivery. We tested the hypothesis using adult bovine articular chondrocytes treated with combinations of cDNAs encoding insulin-like growth factor I, bone morphogenetic protein-2 and protein-7, transforming growth factor β1, and fibroblast growth factor 2. We found that these growth factor transgenes regulated each others growth factor production. This regulation ranged from stimulation to inhibition. Regulation by multiple transgenes was not predictable from the regulatory actions of the individual transgenes. Such interactions may be important for the selection of growth factor genes for cell-based therapies, including articular cartilage repair.

Production of recombinant AAV vectors encoding insulin-like growth factor I is enhanced by interaction among AAV rep regulatory sequences.

BackgroundAdeno-associated virus (AAV) vectors are promising tools for gene therapy. Currently, their potential is limited by difficulties in producing high vector yields with which to generate transgene protein product. AAV vector production depends in part upon the replication (Rep) proteins required for viral replication. We tested the hypothesis that mutations in the start codon and upstream regulatory elements of Rep78/68 in AAV helper plasmids can regulate recombinant AAV (rAAV) vector production. We further tested whether the resulting rAAV vector preparation augments the production of the potentially therapeutic transgene, insulin-like growth factor I (IGF-I).ResultsWe constructed a series of AAV helper plasmids containing different Rep78/68 start codon in combination with different gene regulatory sequences. rAAV vectors carrying the human IGF-I gene were prepared with these vectors and the vector preparations used to transduce HT1080 target cells. We found that the substitution of ATG by ACG in the Rep78/68 start codon in an AAV helper plasmid (pAAV-RC) eliminated Rep78/68 translation, rAAV and IGF-I production. Replacement of the heterologous sequence upstream of Rep78/68 in pAAV-RC with the AAV2 endogenous p5 promoter restored translational activity to the ACG mutant, and restored rAAV and IGF-I production. Insertion of the AAV2 p19 promoter sequence into pAAV-RC in front of the heterologous sequence also enabled ACG to function as a start codon for Rep78/68 translation. The data further indicate that the function of the AAV helper construct (pAAV-RC), that is in current widespread use for rAAV production, may be improved by replacement of its AAV2 unrelated heterologous sequence with the native AAV2 p5 promoter.ConclusionTaken together, the data demonstrate an interplay between the start codon and upstream regulatory sequences in the regulation of Rep78/68 and indicate that selective mutations in Rep78/68 regulatory elements may serve to augment the therapeutic value of rAAV vectors.

Comparison of Efficacy of Endogenous and Exogenous IGF-I in Stimulating Matrix Production in Neonatal and Mature Chondrocytes.

Objective The goal of this study was to compare the efficacy of endogenous upregulation of IGF-I by gene therapy and exogenous addition of insulin-like growth factor I (IGF-I) in enhancing proteoglycan synthesis by skeletally mature and neonatal chondrocytes. Chondrocyte transplantation therapy is a common treatment for focal cartilage lesions, with both mature and neonatal chondrocytes used as a cell source. Additionally, gene therapy strategies to upregulate growth factors such as IGF-I have been proposed to augment chondrocyte transplantation therapies. Methods Both skeletally mature and neonatal chondrocytes were exposed to either an adeno-associated virus-based plasmid containing the IGF-I gene or exogenous IGF-I. Results Analysis of IGF-I and glycosaminoglycan production using a 4-parameter dose-response model established a clear connection between the amount of IGF-I produced by cells and their biosynthetic response. Both neonatal and mature chondrocytes showed this relationship, but the sensitivities were quite different, with EC50 of 0.57 ng/mL for neonatal chondrocytes and EC50 of 8.70 ng/mL IGF-I for skeletally mature chondrocytes. Conclusions These data suggest that IGF-I gene therapy may be more effective with younger cell sources. Both cell types were less sensitive to exogenous IGF-I than endogenous IGF-I.

Human IGF-I propeptide A promotes articular chondrocyte biosynthesis and employs glycosylation-dependent heparin binding

BACKGROUND Insulin-like growth factor I (IGF-I) is a key regulator of chondrogenesis, but its therapeutic application to articular cartilage damage is limited by rapid elimination from the repair site. The human IGF-I gene gives rise to three IGF-I propeptides (proIGF-IA, proIGF-IB and proIGF-IC) that are cleaved to create mature IGF-I. In this study, we elucidate the processing of IGF-I precursors by articular chondrocytes, and test the hypotheses that proIGF-I isoforms bind to heparin and regulate articular chondrocyte biosynthesis. METHODS Human IGF-I propeptides and mutants were overexpressed in bovine articular chondrocytes. IGF-I products were characterized by ELISA, western blot and FPLC using a heparin column. The biosynthetic activity of IGF-I products on articular chondrocytes was assayed for DNA and glycosaminoglycan that the cells produced. RESULTS Secreted IGF-I propeptides stimulated articular chondrocyte biosynthetic activity to the same degree as mature IGF-I. Of the three IGF-I propeptides, only one, proIGF-IA, strongly bound to heparin. Interestingly, heparin binding of proIGF-IA depended on N-glycosylation at Asn92 in the EA peptide. To our knowledge, this is the first demonstration that N-glycosylation determines the binding of a heparin-binding protein to heparin. CONCLUSION The biosynthetic and heparin binding abilities of proIGF-IA, coupled with its generation of IGF-I, suggest that proIGF-IA may have therapeutic value for articular cartilage repair. GENERAL SIGNIFICANCE These data identify human pro-insulin-like growth factor IA as a bifunctional protein. Its combined ability to bind heparin and augment chondrocyte biosynthesis makes it a promising therapeutic agent for cartilage damage due to trauma and osteoarthritis.