标题: Temporal Expression Patterns and Corresponding Protein Inductions of Early Respo [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:49 标题: Temporal Expression Patterns and Corresponding Protein Inductions of Early Respo
Research Service and Geriatrics Research, Education, and Clinical Center, Veterans Affairs Medical Center, Miami, Florida, USA; Department of Biomedical Engineering, University of Miami, Coral Gables, Florida, USA$ J! L6 T& v' g6 N( ~$ V
7 K8 q5 J+ ^6 W9 a- C3 `' o: a5 B; M6 XKey Words. Chondrogenesis ? Adult bone marrow stem cells ? TGF-? receptor ? TGF-?1 ? Gene expression ? Mesenchymal stem cell ; ?* ` R6 u; F) i0 ~/ u 9 G8 E: B* b& _" H+ F- QCorrespondence: Herman S. Cheung, Ph.D., Research Service, Miami VA Medical Center, 1201 NW 16th Street, Miami, Florida 33125, USA. Telephone: 305-575-3388; Fax: 305-575-3365; e-mail: hcheung@med.miami.edu U; d" G1 o8 T
7 v* _; y- Y5 E' s5 ?8 HABSTRACT& ~2 w ~5 @( Z" Q1 ~
8 G3 P; C# ]! C" V# }When damage on articular cartilage extends into the subchondral bone, mesenchymal stem cells (MSCs) migrate from bone marrow (BM) to the injured area and form a new cartilage-like reparative tissue . This clinical finding indicates that local stimuli at the injured site of articular cartilage can induce chondrogenic differentiation of MSCs. It has been demonstrated that continuous passive motion enhanced the repair of full-thickness defects of articular cartilage with autogenous periosteal grafts containing MSCs . A recent study repaired large, full-thickness cartilage defects of rabbit keen joints by transplanting BM-derived MSCs (BM-MSCs) and showed that 6 months after the implantation, different local mechanical environments resulted in substantial differences in mechanical properties of reparative tissues on the posterior and anterior aspects of the repair area . These cartilage repair studies suggest that mechanical stimuli may affect chondrogenic differentiation of MSCs and/or cartilage-specific matrix formation of differentiated cells (i.e., chondrocyte). b4 W Z' J/ l4 Y+ M J
, `. E: r% k# g( P9 NIt has been well documented that compressive loading modulated the cartilage-specific macromolecule biosynthesis of mature chondrocytes , whereas compressive loading was shown to stimulate chondrogenic differentiation of chick and mouse embryonic mesenchymal cells . Recently, Angele et al. showed that cyclic hydrostatic pressure enhanced the extracellular matrix deposition of human BM-MSCs, which underwent chondrogenesis in pellet cultures . Our recent study found that cyclic compressive loading promoted gene expressions of chondrogenic markers (collagen type II and aggrecan) and transforming growth factor-?1 (TGF-?1) in rabbit BM-MSCs in a serum-free media, suggesting that the TGF-? signal pathway may be involved in BM-MSC chondrogenic differentiation stimulated by dynamic compressive loading .% q" N( a K6 b. d6 k2 H% J
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In vitro studies have demonstrated that TGF-? can induce chondrogenesis of BM-MSCs . TGF-? regulates a variety of cellular functions by signaling through a heteromeric receptor complex of two transmembrane serinethreonine kinases (receptor types I and II). The intracellular TGF-? signal transduction is initiated by type I receptor (T?R-I) after its phosphorylation by ligand-bound type II receptor (T?R-II) . Mizuta et al. found that the gene expressions of both receptors and TGF-?1 ligand were upregulated during periosteal chondrogenesis induced by exogenous TGF-?1 treatment wherein the temporal and spatial gene expression patterns of both receptors were consistent with those of the ligand itself , suggesting that TGF-?1 regulated periosteal chondrogenesis. Therefore, the presence of both receptors is essential for chondrogenesis of BM-MSCs when TGF-? signaling is involved.( j+ X! v9 ^' J8 j# v0 w
6 s2 i" d) V' Z3 FActivating protein 1 (AP-1), a transcription factor complex of the Jun and Fos nuclear oncoproteins, is one of the downstream targets for mitogen-activated protein kinase (MAPK) signaling pathway. It has been shown that MAPKs regulated chondrogenesis of chick embryo limb bud cells at postprecartilage condensation stages , whereas overexpression of c-Fos gene inhibited chondrocyte differentiation of ATDC5 chondrogenic cells . Recent studies demonstrated that AP-1 binding activity is a requisite for promoting chondrogenesis of C3H10T1/2 mesenchymal cells . In addition, Kim et al. showed that induction of endogenous TGF-?1 gene expression by TGF-?1 treatment was mediated by binding of the AP-1 complex to the promoter region of TGF-?1 gene, whereas both components (c-Fos and c-Jun) of the AP-1 complex were required for TGF-?1 autoinduction . Therefore, it is possible that activity of AP-1 may regulate BM-MSC chondrogenesis induced by TGF-?.4 J% z" ?6 a% m8 u+ A
# v( T* l1 S5 jSox9, a member of Sry-type high-mobility-group box (Sox) genes, is identified as an essential transcription factor for chondrogenesis of mesenchymal cells and expressed in precartilaginous mesenchymal condensation and maturing cartilage . It has been indicated that Sox9 can regulate expression of chondrocyte-specific collagen (i.e., type II, IX, and XI collagens) and aggrecan genes. Sox9 was able to promote type II collagen gene expression by binding directly to an enhancer element in the first intron of the collagen II gene while it could bind to the promoter of type IX and XI collagen genes . Sox9 also enhanced the gene promoter activity of aggrecan in the TC6 chondrocytic cell line . Recently, it has been demonstrated that overexpression of Sox9 gene in mouse BM-MSCs promoted chondrogenesis in in vitro micromass culture and in vivo transplantation . Because of its capability to regulate chondrogenic gene expressions, Sox9 may play an important role in regulation of chondrocyte differentiation of BM-MSCs. 9 u( g. n& K! v; R" h9 c. O1 h( h" E
Our recent study suggested that dynamic compressive loading might promote chondrogenesis of rabbit BM-MSCs through the TGF-? signaling pathway . To advance our understanding of the mechanism behind this finding, our first step was to examine expressions of the early responsive genes that may regulate chondrogenesis and TGF-? signal transduction. Therefore, the objective of this study was to examine the temporal expression patterns of c-Fos, c-Jun, Sox9, TGF-?1, and TGF-? receptors and induction of their corresponding proteins in agarose cultures of rabbit BM-MSCs under cyclic compressive loading.! K4 W0 Z; e2 G# V6 B( o# }& r% C/ S
* e( K3 a1 r& K) G0 O; y1 yMATERIALS AND METHODS ~+ K5 M& V& _0 X; q9 i' a, u" }+ l
Typical Gene Expressions : K0 |, {0 b! V# F% n+ {6 Z+ a: _$ Z9 v4 p# g
In general, the gene expressions of c-Fos, c-Jun, Sox9, type II collagen, TGF-?1, and T?R-I and T?R-II were upregulated in the samples under dynamic compression, whereas the temporal expressions of those genes, except c-Fos, were different between the first and second days of testing (Figs. 1, 2).% w" D' c& E( q T
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Figure 1. RT-PCR analysis of gene expressions of rabbit BM-MSCs on the first day of testing. Gene expressions of the loading group were analyzed after being subjected to 1 (L1), 2 (L2), and 4 (L4) hours of dynamic compressive loading and after 4 (R4), 8 (R8), and 20 (R20) hours of rest following 4-hour dynamic compressive loading. Gene expressions of the control group were analyzed at the beginning of the compression test. Total RNA was isolated and RT-PCR was performed on 1 μg of each sample using primers for c-Fos, c-Jun, Sox9, T?R-I, T?R-II, TGF-?1, collagen II, and GAPDH as shown on the left. Bands shown are representatives of five independent experiments. Abbreviations: BM-MSC, bone-marrow mesenchymal stem cell; RT-PCR, reverse transcription–polymerase chain reaction.4 [2 ?9 X; w; R
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Figure 2. RT-PCR analysis of gene expressions of rabbit BM-MSCs on the second day of testing. Gene expressions of the loading group were analyzed after being subjected to 1 (L1), 2 (L2), and 4 (L4) hours of dynamic compressive loading and after 4 (R4), 8 (R8), and 20 (R20) hours of rest following 4-hour dynamic compressive loading. Gene expressions of the control group were analyzed at the beginning of the compression test. Total RNA was isolated and RT-PCR was performed on 1 μg of each sample using primers for c-Fos, c-Jun, Sox9, T?R-I, T?R-II, TGF-?1, collagen II, and GAPDH as shown on the left. Bands shown are representatives of five independent experiments. Abbreviations: BM-MSC, bone-marrow mesenchymal stem cell; RT-PCR, reverse transcription–polymerase chain reaction. $ V2 e1 y2 b4 J! b, [# ? ' N7 f" A0 @/ }Day 1 of Testing ? During the 4-hour compression test, gene expressions of c-Jun, Sox9, type II collagen, TGF-?1, T?R-I, and T?R-II for the loading group gradually increased and reached the peak after 2 hours of loading and then decreased from that peak after 4 hours of loading. Only the loading group exhibited a weak expression of c-Fos gene after 1 hour of loading (Fig. 1). After the 4-hour compression test, all gene expressions of the loading group decreased to a level similar to (i.e., c-Jun, type II collagen, TGF-?1, and T?R-II) or slightly higher than (i.e., Sox9) those of the control group except that the expression of the T?R-I gene exhibited another peak after 8 hours of rest (Fig. 1).* S' ~4 ^& R0 j# U
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Day 2 of Testing ? During the 4-hour compression test, all gene expressions of the loading group were upregulated. The gene expressions of c-Jun, Sox9, type II collagen, T?R-I, and T?R-II for the loading group gradually increased and reached the peak after 4 hours of loading, whereas the gene expression of TGF-?1 quickly reached the highest level after 1 hour of loading (Fig. 2). Similar to the first day of testing, only weak expression of c-Fos gene was seen for the loading group after 1 hour of loading (Fig. 2). After the 4-hour compression test, gene expressions of c-Jun, Sox9, type II collagen, TGF-?1, and T?R-I for the loading group gradually decreased to a level slightly higher than those of the control group, whereas T?R-I gene expression was similar to those of the control group (Fig. 2).! g( J8 E. a) W* @- b9 ` E/ d
V% d8 Y, s% p# ?# M5 UStatistical Analysis of Gene Expressions# j% s* c) s) |1 ^( M ]0 g
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Day 1 of Testing ? Significant differences were found in all gene expressions between two experimental groups with the samples subjected to 2-hour dynamic compression having a higher level of expression than the control group (Figs. 3–5). After 4-hour dynamic compression, the loading group still exhibited significantly greater expression of type II collagen than the control (Fig. 5). Compared with the control group, the loading group also exhibited significantly higher levels of T?R-I and T?R-II gene expressions after 1-hour dynamic compression as well as higher expression of T?R-I gene after 8 hours of rest following 4 hours of dynamic compression (Fig. 3).5 u' l; G; U# X& w. J, @+ Y- ?
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Figure 3. Statistical analysis of gene expressions of TGF-? type I and type II receptors. The relative gene expressions of TGF-? type I and type II receptors among six time points of 2 days of testing, as presented in Figures 1 and 2, were statistically analyzed using a one-way analysis of variance with Student-Newman-Keuls comparison test (n = 10 for control group; n = 5 for loading group). Gene expressions of the loading group were analyzed after being subjected to 1 (L1), 2 (L2), and 4 (L4) hours of dynamic compressive loading and after 4 (R4), 8 (R8), and 20 (R20) hours of rest following 4-hour dynamic compressive loading on each day of testing. Gene expressions of the control group were analyzed at the beginning of the compression test and the recovery period on each day of testing. ; ~ d5 P" {( w $ J4 H4 ]1 O$ |' Q% t5 V" ?Figure 4. Statistical analysis of gene expressions of TGF–?1 and c-Jun. The relative gene expressions of TGF–?1 and c-Jun among six time points of 2 days of testing, as presented in Figures 1 and 2, were statistically analyzed using a one-way analysis of variance with Student-Newman-Keuls comparison test (n = 10 for control group; n = 5 for loading group). Gene expressions of the loading group were analyzed after being subjected to 1 (L1), 2 (L2), and 4 (L4) hours of dynamic compressive loading and after 4 (R4), 8 (R8), and 20 (R20) hours of rest following 4-hour dynamic compressive loading on each day of testing. Gene expressions of the control group were analyzed at the beginning of the compression test and the recovery period on each day of testing. 5 F! M k1 b* _" ]( [2 [" \ ( b2 s; U0 s3 _2 ]/ e+ uFigure 5. Statistical analysis of gene expressions of Sox9 and type II collagen. The relative gene expressions of Sox9 and type II collagen among six time points of 2 days of testing, as presented in Figures 1 and 2, were statistically analyzed using a one-way analysis of variance with Student-Newman-Keuls comparison test (n = 10 for control group; n = 5 for loading group). Gene expressions of the loading group were analyzed after being subjected to 1 (L1), 2 (L2), and 4 (L4) hours of dynamic compressive loading and after 4 (R4), 8 (R8), and 20 (R20) hours of rest following 4-hour dynamic compressive loading on each day of testing. Gene expressions of the control group were analyzed at the beginning of the compression test and the recovery period on each day of testing. ) G7 R4 p0 h* W5 I4 `( C 7 R3 J( p5 g& FDay 2 of Testing ? Significant differences were found in all gene expressions between two experimental groups with the samples subjected to 4-hour dynamic compression having higher levels of expression than those of the control group (Figs. 3–5). For the gene expression of TGF-?1, the samples subjected to 1- and 2-hour dynamic compression exhibited significantly higher levels of expression than the control group (Fig.4). In addition, samples subjected to 2-hour dynamic compression also exhibited a significantly greater expression of T?R-II gene than the control group (Fig. 3). However, no significant differences were found in all gene expressions between two experimental groups during the recovery period./ `4 Z; }' Z* O3 k8 r& J3 h' [
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Typical Protein Induction 8 c o- i# J( O: @9 V( o; O U! [9 @) A/ X0 v1 b' a+ `In general, the protein inductions of c-Jun, Sox9, TGF-?1, T?R-I, and T?R-II were seen in the samples subjected to 2- and 4-hour dynamic compression (Fig. 6). The protein inductions of c-Jun, Sox9, T?R-I, and T?R-II reached the highest levels in the loading group after 4 hours of loading and then decreased to a level similar to the control group after 4 hours of rest (Fig. 6). However, after the protein induction of TGF-?1 in the loading group by the 2-hour dynamic compression, the protein level of TGF-?1 was maintained at the similar level after 4-hour loading and during the 4-hour recovery period (Fig. 6). " Y. A3 n2 H) D5 {& `) j. ^ ! }' ?" m0 s7 kFigure 6. Western blot analysis of corresponding protein inductions of c-Jun, Sox9, TGF-?1, and TGF-? receptors on the first day of testing. Protein inductions of the loading group were analyzed after being subjected to 2 (L2) and 4 (L4) hours of dynamic compressive loading and after 2 (R2) and 4 (R4) hours of rest following 4-hour dynamic compressive loading. Protein inductions of the control group were analyzed at the beginning of the compression test. Nuclear extract of NIH/3T3 mouse fibroblasts treated with phorbol, cell lysate of rabbit chondrocytes, recombinant protein of mouse T?R-I, fusion protein of human T?R-II, and cell lysate of human normal fibroblasts were used as positive controls ( ) for c-Jun, Sox9, T?R-I, T?R-II, and TGF-?1, respectively. Bands shown are representative of four independent experiments. 1 K2 x3 B5 X6 q" a/ t* `1 h, e0 K! J
DISCUSSION ' T' ^; c; h, M. D$ S , n4 z$ Z2 Y8 A* q; JThis work was supported by a National Institutes of Health grant (AR 38421) and a VA Merit Review Grant. The authors would like to thank Ms. Kristen Hagar for her technical assistance with RT-PCR and cell culture.% [1 S1 I8 k! ^+ t" ~
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