![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 19, Issue 12, 5446-5455, December 2008
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
*Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh, PA 15261; Department of Radiation Oncology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15232; Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390; and Department of Cancer and Thoracic Surgery, Okayama University School of Medicine, Okayama 700-8558, Japan
Submitted March 17, 2008;
Revised September 19, 2008;
Accepted September 30, 2008
Monitoring Editor: Jonathan Chernoff
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Roberts et al., 2003; Siegel and Massague, 2003; Bierie and Moses, 2006a). Deregulation of TGF-β has been implicated in enhancing progression of malignancy (Bierie and Moses, 2006b; Gupta and Massague, 2006). Transduction of TGF-β signals from the cell surface to the nucleus is mediated by dimerization of type II and type I receptors followed by activation of a cascade of intracellular signal-transducing proteins called Smads (Massague and Wotton, 2000). The effects of TGF-β on cell lineage differentiation during development and in tumor cell growth during stages of tumorigenesis are variable (Siegel and Massague, 2003; Waite and Eng, 2003). The downstream TGF-β–activated effectors are quite different based on cell type and cellular status (Roberts et al., 2003; Siegel and Massague, 2003). The prominent function of TGF-β in tumor suppression as well as cellular differentiation is through cell cycle regulation (Liu, 2006). Various TGF-β–regulated molecules have been identified with functions that principally govern G1 progression (Massague, 2004; Liu, 2006), whereas some mitotic players, including cyclin B and CDC2, are also implicated as targets of TGF-β signaling during the cell cycle (Choi et al., 1999; Liu et al., 1999; Wan et al., 2001). Recent studies addressing Smad3 function in cells of the hematopoietic system have revealed that knockout of Smad3 in mice results in a significant accumulation of a G2/M population of bone marrow stromal cells (BMSCs) (Epperly et al., 2006). This observation suggested a potential novel regulatory mechanism for TGF-β in G2/M progression during the cell cycle. The mechanism of such a G2/M regulation by TGF-β signaling pathway is unknown.
In tumor development, mutated stromal cells can alter the tumor microenvironment and promote metastasis (Wiseman and Werb, 2002; De Wever and Mareel, 2003; Orimo et al., 2005; Dong and Blobe, 2006; Roberts et al., 2006). In the hematopoietic system, BMSCs are an integral component of the bone marrow that provides and responds to cytokines that are involved in directing cell lineage (Pittenger et al., 1999). Deregulation of TGF-β signaling in the stroma affects immune cells involved in micro-immunosurveillance and thereby also can promote migration by tumor cells (De Wever and Mareel, 2003; Epperly et al., 2005; Dong and Blobe, 2006). The expression of TGF-β in BMSCs contributes to the development of the hematopoietic cell lineages, including bone, cartilage, fat, tendon, and muscle (Pittenger et al., 1999). Disruption of TGF-β signal transduction in BMSCs can lead to alteration of cell cycle progression, cell migration, and to radioresistance, which in turn can affect hematopoiesis as well as bone marrow-mediated tumorigenesis (Epperly et al., 2005, 2006; Zhou et al., 2005). Previous studies have focused extensively on how TGF-β signaling integrates with other signaling pathways to regulate cell lineage differentiation and cell migration and adhesion (Watanabe and Whitman, 1999; Massague and Chen, 2000; Waite and Eng, 2003). Little is known about the role of TGF-β in regulating cell cycle progression in BMSCs. The recent discovery that loss of TGF-β signaling abrogates BMSC G2/M progression suggests a new direction for studying TGF-β function. Understanding the molecular basis of how G2/M is regulated by TGF-β could unveil a novel mechanism by which TGF-β affects G2/M during the cell cycle and further enhance our knowledge of stromal cell biology.
To elucidate the mechanism by which G2/M is regulated by TGF-β signaling in BMSCs, we have integrated biochemical and immunocytochemical assays with an in vivo analysis. We demonstrate that the accumulation of cells at G2/M in Smad3–/– cells is attributable to a failure of TGF-β–mediated mitotic regulation. In addition, we show that regulation of mitotic progression by TGF-β in BMSCs is through targeting the APC–separase pathway. Together, these results suggest that APC-dependent regulation of CDK1 activity, which in turn modulates chromatid separation, is a target for TGF-β signaling during mitosis.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Bone marrow stromal cells were cultured in 
-minimal essential medium (MEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS) (Atlanta Biologicals, Norcross, GA), 0.2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). Primary bone marrow stromal cells were grown in MyeloCult 5300 (Stem Cell Technologies, Vancouver, BC, Canada). To synchronize cells in mitosis, cells were grown in the presence of 0.4 mM thymidine (Sigma-Aldrich, St. Louis, MO) for 18 h, washed with phosphate-buffered saline (PBS), and grown in the fresh medium for 4 h, followed by 50 ng/ml nocodazole for 12 h and release (Wan et al., 2001). To synchronize cells at the G1/S transition, cells were treated with thymidine twice and then released. For flow cytometric analysis, cells fixed in ethanol were washed and subsequently stained in PI buffer [10 µg/ml propidium iodide, 5 µg/ml RNase A [Sigma-Aldrich] in PBS) (Wan et al., 2001). Transfection was done in Opti-MEM1 (Invitrogen) using Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. Infection/transduction of retrovirus carried Smad3 in Smad3–/– cells was described previously (Liu et al., 2000).
Mutagenesis
Mutants, including separase (S1073A/S1126A/S1305A/T1346A/S1501A/S1508A/S1545A/S1552A), separase (S1073A/S1305A/T1346A/S1501A/S1508A/S1545AS1552A), and separase (S1126A), were engineered using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The presence of the desired substitutions and the absence of other mutations were confirmed by sequence.
Antibodies and Reagents
Immunoblotting analyses and immunoprecipitation were performed using the following antibodies: Cdh1 (Calbiochem, San Diego, CA), CDC2 (Santa Cruz Biotechnology, Santa Cruz, CA), Plk (Santa Cruz Biotechnology), BubR1 (Santa Cruz Biotechnology), NEK (Santa Cruz Biotechnology), securin (MBL International, Woburn, MA), cyclin B1 (Santa Cruz Biotechnology), APC2 (Santa Cruz Biotechnology), APC6 (Santa Cruz Biotechnology), separase (Santa Cruz Biotechnology; Novus Biologicals, Littleton, CO), tubulin (Calbiochem), and horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit and anti-rat secondary antibody (Promega, Madison, WI). Monoclonal anti-TGF-β neutralizing antibody (R&D Systems, Minneapolis, MN) was used for TGF-β blocking experiment. Western blot analysis was performed using ECL detection kit (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). Semiquantification of data was performed using densitometer.
Ubiquitylation Assay
Wild-type and Smad3 deletion cells were synchronized using nocodazole, and cells were subsequently collected at four time points (1, 2, 3, and 4 h after release) followed by mixing four populations. Cell lysates were prepared following a hypotonic method described previously (Wan et al., 2001). APC was purified using an antibody against Cdc27. APC were subsequently subjected to a ubiquitination cocktail (50 µg/ml Ubc5, 1.25 mg/ml ubiquitin, 200 µg/ml recombinant E1, 0.1 mg/ml cycloheximide, and 2 µM ubiquitin aldehyde) with 1/10 the volume of 35S-labeled in vitro-translated cyclin B. Ubiquitination was reflected in the formation of high-molecular polyubiquitin conjugates. Smeared ubiquitin conjugates were measured by densimeter.
H1 Kinase Assay
Cells were lysed with lysis buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol [DTT] 1 mM EDTA, 0.2% Nonidet P-40, and 5 mM NaF, supplemented with protease inhibitor cocktails). Lysates were incubated with 2 µg of anti-cyclin B1 antibody (Santa Cruz Biotechnology) overnight at 4°C. Thereafter, protein A/G beads were added to the lysate and incubated for additional 2 h. Immunoprecipitation (IP) complex was washed three times with lysis buffer and once with HBS (10 mM HEPES, pH 7.4, and 150 mM NaCl) before adding the kinase buffer (5 µg of histone H1 in 20 µl of HBS containing 15 mM MgCl2, 50 µM ATP, 1 mM DTT, and 10 µCi of [
-32P]ATP) for 10 and 30 min at 30°C. Sample buffer was added to stop the reaction and 20 µl of each sample was analyzed in 4–12% SDS-polyacrylamide gel electrophoresis (PAGE), followed by autoradiography (Stemmann et al., 2001).
32P-Labeling In Vivo
Cells were incubated in phosphate-free DMEM (Invitrogen) containing 10% dialyzed FCS (Invitrogen). Thereafter, cells were exposed for 1 h in [32P]orthophosphate (PerkinElmer Life and Analytical Sciences, Boston, MA) at a final activity of 0.4 mCi/ml in phosphate-free DMEM. Then, cells were harvested in IP lysis buffer (50 mM Tris-HCl, pH 7.7, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 1 mM DTT, 2 mM MgCl2, 1 µM okadaic acid, and 1 µM microcystin LR, supplemented with protease inhibitor cocktail). Cell lysates were incubated with 5 µg of anti-CDC27 antibody (Santa Cruz Biotechnology) or 2 µg of anti-separase antibody (Novus Biologicals) followed by incubation with protein A/G beads (Pierce Chemical. Rockford, IL). Immunoprecipitated samples were washed once with lysis buffer and three times with wash buffer (20 mM Tris-HCl, pH 7.7, 0.2% NP-40, 1 mM EDTA, 2 mM MgCl2, 1 µM okadaic acid, and 1 µM microcystin LR). Samples were resolved using SDS-PAGE followed by autoradiography (Bakkenist and Kastan, 2003).
Immunoprecipitation
Cells were lysed with lysis buffer (for Cdh1 and Cdc20: 10 mM Tris-HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2 mM, phenylmethylsulfonyl fluoride, 0.5% NP-40, 0.1% SDS, and protease inhibitor cocktail; and for Cdc27: 50 mM Tris-HCl, pH 7.7, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 1 mM DTT, 2 mM MgCl2, 1 µM okadaic acid, and 1 µM microcystin LR, supplemented with protease inhibitor cocktail), and lysate was incubated with 5 µg of anti-CDC27 antibody (Santa Cruz Biotechnology) followed by incubation with protein A/G beads (Pierce Chemical). Immunoprecipitated samples were washed and analyzed by SDS-PAGE. Western blot was then performed using anti-CDC27 (Santa Cruz Biotechnology), anti-Cdh1 (Calbiochem), and anti-CDC20 antibody (Liu et al., 2007).
Immunofluorescence Microscopy
Cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100. Immunofluorescent analysis was performed using anti-tubulin (Calbiochem), anti-phospho-smad3 (Cell Signaling Technology, Danvers, MA), and anti-phospho-histone 3 (Millipore, Billerica, MA). Secondary antibodies were Cy2, fluorescein isothiocyanate (Jackson ImmunoResearch Laboratories, West Grove, PA), and Texas-Red (Jackson ImmunoResearch). For analysis of mitotic cells, number of phospho-histone H3-positive cells was counted among 1000 cells in different fields at least three times (Tang et al., 2006).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Roberts et al., 2006). Previous studies have extensively examined the mechanism by which the cell cycle is regulated by TGF-β signaling, in which a number of critical G1 regulators, such as p15, p21, p27, CDK4.6, and CDK2, are regulated by TGF-β in various types of epithelial cells (Datto et al., 1995; Massague and Chen, 2000; Liu, 2006). Recent studies on stromal cells have sketched the basic framework of TGF-β affecting tumor microenvironment, resulting in subsequent metastasis (De Wever and Mareel, 2003; Dong and Blobe, 2006). To explore the cellular function of TGF-β in stromal cells, we have established Smad3-deficient BMSCs and then dissected the effect of TGF-β on cell cycle progression (Figure 1A).
|
5% of wild-type BMSCs stained positively for phospho-histone H3 cells (Figure 1C). To validate the notion that Smad3 modulates mitotic progression, we collected bone barrow from Smad3–/– mouse and established primary bone marrow stromal cells for mitotic analysis using phospho-histone H3 as readout. As indicated in Figure 1D, 15% of the primary stromal cells were in mitosis for Smad3–/– mouse, whereas 5% of wild-type stromal cells were mitosis. In summary, these results suggest that Smad3 mediated TGF-β signaling modulates mitotic progression in bone marrow stromal cells. Accumulation of the mitotic population due to an incomplete TGF-β signaling pattern could probably have resulted from failure in the regulation of chromatid separation.
|
). To examine the effect of TGF-β signaling in the regulation of mitosis, we determined the effect of the presence of activated Smad3 on mitosis by biochemical and immunocytochemical analyses. We first measured the TGF-β response in wild-type or Smad3–/– bone marrow stromal cells by using phospho-Smad3 as readout. As shown in Figure 2A, a basal TGF-β response was detected in wild-type cells, suggesting that basal TGF-β1 is secreted to the cultured medium, which ensures cellular metabolism. Administration of cells with TGF-β1 at 100 pM significantly increased the activation of TGF-β pathway, whereas no phospho-Smad3 was detected in the Smad3 deletion cells (Figure 2A). Neutralization of TGF-β ligand by excess supplementation of antibody against TGF-β1 significantly blocked the activation of TGF-β signaling in the wild-type cells (Figure 2A). To test the status of TGF-β signaling during the cell cycle, we synchronized wild-type BMSCs with a double thymidine block. Cells were then released and collected at different time points for immunoblotting and fluorescence-activated cell sorting (FACS) analyses as indicated (Figure 2B). The result shown in Figure 2B of phosphorylation of Smad3 was measured over a broad range during the cell cycle. Activation of TGF-β as reflected by phospho-Smad3 is detected during the mitosis (Figure 2B). To validate the result by immunoblotting, we analyzed the localization of phosphorylated Smad3 in mitotic cells by using an immunocytochemical analysis. As shown in Figure 2C, activated Smad3 was detected in anaphase as well as telophase in wild-type cells, whereas no activated Smad3 was found in metaphase chromosomes in Smad3–/– cells. This result further suggests the role of TGF-β involved in mitotic regulation.
The APC Pathway Is Affected by Deletion of Smad3
To obtain insight into the mitotic apparatus, which mediates the TGF-β response, we systematically analyzed the critical molecules that are involved in mitotic progression (Barr et al., 2004). We conducted an analysis of the protein expression profile of a cluster of mitotic regulators as indicated (Figure 3, A and B). The relative abundance of candidate proteins was measured using immunoblotting, comparing expression in wild-type and Smad3–/– cells. As shown in Figure 3, A and B, the expression of several proteins (phosphorylation and protein levels) exhibited dramatic differences between wild-type and Smad3–/– cells. Among the tested proteins, phosphorylation of CDC27 (reflected by the protein shift) in wild-type cells was significantly stronger than that of CDC27 in Smad3–/– cells. In contrast, phosphorylation of separase (represented by the protein shift) in Smad3–/– cells is much stronger than that of separase in wild-type cells (Figure 3, A and B). Meanwhile, we also noticed the elevated expression of cyclin B in Smad3–/– cells but not in the wild-type cells. Moreover, a smaller amount of separase cleavage products (C terminus) was measured in Smad3–/– cells than wild-type cells. Given the key function of the APC in governing chromatid separation pathway (Stemmann et al., 2001; Holland and Taylor, 2006), the protein expression profiling suggests that the APC-separation pathway may be the site in the molecular cascade that mediates the TGF-β effect on mitosis.
|
). As shown in Figure 4A, intense phosphorylation of CDC27 (protein band is shifted and smeared) was detected in wild-type cells, whereas weaker CDC27 phosphorylation was observed in Smad3–/– cells. Consistent with IP immunoblotting experiment, incorporation of radiolabeled phosphate in wild-type cells is about fourfold stronger than that in Smad3–/– cells (Figure 4, B and D). Rescue of Smad3 restored CDC27 phosphorylation as indicated in Figure 4, C and D. This result further suggests that the TGF-β effect in mitosis is to regulate CDC27 through phosphorylation.
|
4 h compared with wild-type cells. These results suggest that TGF-β–mediated alteration of cyclin B could affect mitotic progression.
|
Given the data showing that TGF-β affects CDC27 phosphorylation and that TGF-β alters cyclin B protein levels, we next asked whether TGF-β–mediated modulation of CDC27 phosphorylation regulated APC activity in the destruction of cyclin B (Choi et al., 1999; Wan et al., 2001). We purified APC from wild-type or Smad3–/– cells during mitosis and analyzed its activity using 35S-labeled and in vitro-synthesized cyclin B1 as a putative substrate (Fang et al., 1998). As shown in Figure 5, C and D, targeted deletion of Smad3 in BMSC cells significantly altered APC activity for ubiquitylating cyclin B. Given that cyclin B is an important component of CDK1, we further measured the effect of TGF-β on CDK1 by using a H1 kinase assay (Stemmann et al., 2001). Consistent with the result from the analysis of APC activity, CDK1 activity is significantly higher in Smad3–/– cells than in wild-type cells as reflected by the different levels of cyclin B protein (Figure 5, E and F). Restoration of Smad3 rescued APC activity and therefore reduced CDK1 activity to approximately normal levels (Figure 5, D and E).
Uncontrolled CDK1 Suppresses Chromatid Separation via Phosphorylation of Separase
Comparison of protein profiling has shown a significant difference in separase phosphorylation between wild-type and Smad3–/– cells (as reflected by shift of the full-length separase band). Although phosphorylated separase is maintained at high levels in Smad3–/– cells, the production of cleaved C-terminal separase is significantly decreased (Figure 6A). To confirm the effect of TGF-β on the status of separase phosphorylation, we examined the capacity of phosphate incorporation of separase in BMSCs in the presence or absence of Smad3 (Bakkenist and Kastan, 2003). As shown in Figure 6, B and C, disruption of Smad3 lead to a significant increase in phosphate incorporation for separase. Previous studies have suggested that uncontrolled CDK1 activity in mitosis could affect the activation of separase through phosphorylation (Stemmann et al., 2001; Holland and Taylor, 2006). Our results from dissection of the role of Smad3 in mitotic regulation demonstrated that targeted deletion of Smad3 results in down-regulation of CDC27 phosphorylation, which in turn leads to accumulation of cyclin B, thereby elevating CDK1 activity (Figures 3
–5). Together, these results suggested a regulatory-axis of TGF-β–modulating chromatid separation through the TGF-β–Smad3–APC–CDK1–separase pathway.
|
). To map the phosphorylation site on separase targeted by CDK1 initiated through TGF-β signaling, we have expressed various mutated separases in Smad3–/– cells, including Ser1073, Ser1126, Ser1305, Thr1346, Ser1501, Ser1508, Ser1545, and Ser1552, and subsequently measured the incorporation of phosphate into separase. As shown in Figure 6, D–F, replacing Ser by Ala at 1126 in separase significantly abolished phosphorylation of separase. Expression of separase mutant (S1126A) in Smad3–/– cells significantly restored the Smad3–/– phenotype (Supplemental Figure 2), suggesting that Ser1126 is critical for mediating TGF-β–induced phosphorylation of separase. The results from these experiments define a novel regulatory mechanism by which TGF-β signaling modulates mitotic progression through effects on the Cdh1–APC–CDK1–separase cascade (Figure 7).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Gupta and Massague, 2006; Roberts et al., 2006). In this study, we have identified a new role for TGF-β in modulating mitosis in bone marrow stromal cells. Dysfunction in TGF-β signaling through targeted disruption of Smad3 causes significant delay in mitotic progression. APC, a master mitotic regulator, is targeted by TGF-β signaling for regulation. Regulation of APC by TGF-β results in the control of CDK1 activity. Uncontrolled elevated CDK1 inhibits chromatid separation via phosphorylation of separase. Demonstration of TGF-β–mediated mitotic regulation unravels a previous mystery in which mitotic regulator is down-regulated in response to TGF-β signaling (Choi et al., 1999; Wan et al., 2001) and further explains the presence of activated Smad2/3 detected during mitosis (Batut et al., 2007), which advances our understanding of TGF-β biology.
Prior studies generally focused on epithelial cells have established a paradigm for TGF-β in the regulation of cell cycle. Several critical cell cycle regulators such as p15, p21, p27, CDK4/6, and CDK2 have been shown to respond to TGF-β signaling, suggesting that the G1 phase serves as a window mediating the TGF-β effect during the cell cycle (Datto et al., 1995; Massague and Chen, 2000; Liu, 2006). Recent studies on stromal-epithelial interaction have greatly attracted attention to the role of TGF-β in stromal cells due to its importance in orchestrating the microenvironment eliciting onset of tumor malignancy (De Wever and Mareel, 2003; Epperly et al., 2005; Dong and Blobe, 2006). The mitotic phenotype in the absence of Smad3 in hematopoietic stromal cells is not the same as in MEF cells, suggesting that the cellular behavior of BMSCs in response to TGF-β might not be identical to other types of cells. Results from this work suggest that TGF-β is crucial to ensure normal mitotic progression through modulating the APC–chromatid separation pathway. Given that mitosis is a critical step for segregation of two daughter genomes with high fidelity, coordination of multiple pivotal events during mitosis, such as the mitotic checkpoint and chromatid segregation, needs a sophisticated regulatory system that can ensure precise mitotic progression with a regulatory circuitry on multiple levels. Both failure to control chromatid separation in stromal cells and deregulation of stromal cell cycle due to failure in TGF-β signaling could affect hematopoiesis resulting in tumor metastasis. Dissecting the role of TGF-β in the modulation of mitotic progression in BMSCs has enabled the expansion of the biological function of TGF-β and further enhanced our understanding of hematological malignancy.
TGF-β Modulates Activity of APC via Regulating CDC27 Phosphorylation
APC has been shown to be a critical regulator bridging various environment signals, including genotoxic stress, mitogenic signaling, and TGF-β that induce downstream cellular response (Wan et al., 2001; Chabes et al., 2003; Palframan et al., 2006). Regulation of APC could be achieved at several layers, including phosphorylation of APC, CDC20/Cdh1-activators of APC, and Emi1/CDC14-regulators of CDC20 or Cdh1 (Reimann et al., 2001; Vodermaier, 2004). Phosphorylation of CDC27 was thought to be crucial for activation of APC in mitosis (King et al., 1995; Kraft et al., 2003). Results based on the analyses of Cdc27 phosphorylation, cyclin turnover, measurement of APC activity, and immunocytochemical description of mitotic progression in both wild-type and Smad3–/– cells suggested the conclusion that the TGF-β/Smad3–involved mitotic regulation is mediated by regulating APC activity via alteration of Cdc27 phosphorylation (Supplemental Figure 4). Data from the Mad2 depletion experimental suggested that TGF-β/Smad3-involved mitotic regulation is independent from the spindle assembly checkpoint (Supplemental Figure 3). Our data further suggest the presence of an unknown kinase, which mediates the Smad3 transmitted TGF-β signaling for phosphorylation of CDC27. The consequence of TGF-β–induced phosphorylation of CDC27 results in greater affinity between the activator-Cdh1 and CDC27, thereby achieving activation of APC. Given the presence of multiple phosphorylation sites on CDC27, we do not know the TGF-β responsive phosphorylation site in CDC27 at this time. Identifying the kinase mediating the TGF-β-induced CDC27 phosphorylation and determining the phosphorylation site on CDC27 are the aims of further study.
Alteration of CDK1 by TGF-β Results in Regulating the Chromatid Separation
One of major functions of APC in mitosis is to govern cyclin B protein levels through proteolysis (Glotzer et al., 1991). Cyclin B levels need to be down-regulated in late mitosis enabling lower CDK1 activity, which promotes chromatid separation and exit from mitosis (Stemmann et al., 2001). Previous studies have demonstrated that high CDK1 activity during the mitosis inhibits chromatid separation via phosphorylation of separase (Stemmann et al., 2001; Holland and Taylor, 2006). Among eight mitotic phosphorylation sites in separase, our results have shown that Ser 1126 mediates phosphorylation catalyzed by CDK1, which is consistent with the previous observation (Stemmann et al., 2001; Holland and Taylor, 2006). To confirm the critical role of Ser 1126 in TGF-β regulated chromatid separation, a dominant-negative separase could be engineered and knock-in analysis of Ser 1126 would be desirable to elucidate the mechanism by which separase is regulated through TGF-β–APC–CDK1 cascade. Demonstration that chromatid separation modulated by TGF-β–initiated alteration of CDK1 has revealed a new pathway for the control of chromatid separation.
Recent work suggested the role of APC/Cdc20 in degradation of p21 for G2/M transition (Amador et al., 2007). Given p21 as one of the major downstream of TGF-β signaling, we also asked whether the p21 cold be the site for the TGF-β–mediated mitotic regulation. Results based on monitoring p21 kinetics in mitosis and G1 between wild-type and Smad3 knockout cells further confirmed that CDK1 is the target for TGF-β signaling in modulating mitosis (Supplemental Figure 1, A and B).
Possible Role of the Regulation of the Bone Marrow Stromal Cell Cycle by TGF-β in Tumorigenesis
The cellular microenvironment is governed by stromal cells and is crucial for the maintenance of cell function and tissue integrity. An aberrant microenvironmental biology caused by dysfunctional stromal cells could alter the fate of adjacent cells and possibly could promote malignancy (Epperly et al., 2005, 2006; Zhou et al., 2005). In the hematopoietic system, bone marrow stromal cells are integral components of hematopoiesis, and they secrete and respond to cytokines, provide adhesion elements for hematopoietic cells, and create stem cell homing. Regulation of stromal cellular function by TGF-β has been shown to be under the control of the hematopoietic cell lineage. Uncontrolled TGF-β signaling in bone marrow stromal cell alters its cellular properties, including proliferation and cell adhesion, which in turn stimulate secretion of growth factors, thereby enhancing tumor growth and metastasis (Epperly et al., 2005, 2006; Zhou et al., 2005). The present study implicates basal levels for TGF-β signaling as critical to ensure a normal cell cycle for BMSCs, especially mitotic progression. Results from most of the previous studies based on epithelial cells have attracted much attention to the cytostatic effect of TGF-β on regulation G1. Thorough evaluation of TGF-β effects on cell cycle regulation in synchronized cells has not been reported. Whether the TGF-β–mediated mitotic response is solely stromal cell dependent remains unknown. The precise physiological consequence of TGF-β–mediated mitotic regulation in marrow stromal cells is still unknown. Given the critical role of stromal cells in hematopoiesis, the information provided from the present study may be of value in furthering our knowledge of stromal cell biology.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Address correspondence to: Yong Wan (yow4{at}pitt.edu).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bakkenist, C. J., and Kastan, M. B. (2003). DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421, 499–506.[CrossRef][Medline]
Barr, F. A., Sillje, H. H., and Nigg, E. A. (2004). Polo-like kinases and the orchestration of cell division. Nat. Rev. Mol. Cell Biol 5, 429–440.[CrossRef][Medline]
Batut, J., Howell, M., and Hill, C. S. (2007). Kinesin-mediated transport of smad2 is required for signaling in response to TGF-beta ligands. Dev. Cell 12, 261–274.[CrossRef][Medline]
Bierie, B., and Moses, H. L. (2006a). TGF-beta and cancer. Cytokine Growth Factor Rev 17, 29–40.[CrossRef][Medline]
Bierie, B., and Moses, H. L. (2006b). Tumour microenvironment: TGFbeta: the molecular Jekyll and Hyde of cancer. Nat. Rev. Cancer 6, 506–520.[CrossRef][Medline]
Chabes, A. L., Pfleger, C. M., Kirschner, M. W., and Thelander, L. (2003). Mouse ribonucleotide reductase R2 protein: a new target for anaphase-promoting complex-Cdh1-mediated proteolysis. Proc. Natl. Acad. Sci. USA 100, 3925–3929.
Choi, K. S., Eom, Y. W., Kang, Y., Ha, M. J., Rhee, H., Yoon, J. W., and Kim, S. J. (1999). Cdc2 and Cdk2 kinase activated by transforming growth factor-β1 trigger apoptosis through the phosphorylation of retinoblastoma protein in FaO hepatoma cells. J. Biol. Chem 274, 31775–31783.
Datto, M. B., Li, Y., Panus, J. F., Howe, D. J., Xiong, Y., and Wang, X. F. (1995). Transforming growth factor beta induces the cyclin-dependent kinase inhibitor p21 through a p53-independent mechanism. Proc. Natl. Acad. Sci. USA 92, 5545–5549.
De Wever, O., and Mareel, M. (2003). Role of tissue stroma in cancer cell invasion. J. Pathol 200, 429–447.[CrossRef][Medline]
Derynck, R., Akhurst, R. J., and Balmain, A. (2001). TGF-beta signaling in tumor suppression and cancer progression. Nat. Genet 29, 117–129.[CrossRef][Medline]
Dong, M., and Blobe, G. C. (2006). Role of transforming growth factor-beta in hematologic malignancies. Blood 107, 4589–4596.
Epperly, M. W., Cao, S., Goff, J., Shields, D., Zhou, S., Glowacki, J., and Greenberger, J. S. (2005). Increased longevity of hematopoiesis in continuous bone marrow cultures and adipocytogenesis in marrow stromal cells derived from Smad3(–/–) mice. Exp. Hematol 33, 353–362.[CrossRef][Medline]
Epperly, M. W., Goff, J. P., Zhang, X., Niu, Y., Shields, D. S., Wang, H., Shen, H., Franicola, D., Bahnson, A. B., Nie, S., Greenberger, E. E., and Greenberger, J. S. (2006). Increased radioresistance, g(2)/m checkpoint inhibition, and impaired migration of bone marrow stromal cell lines derived from Smad3(–/–) mice. Radiat. Res 165, 671–677.[CrossRef][Medline]
Fang, G., Yu, H., and Kirschner, M. W. (1998). Direct binding of CDC20 protein family members activates the anaphase-promoting complex in mitosis and G1. Mol. Cell 2, 163–171.[CrossRef][Medline]
Glotzer, M., Murray, A. W., and Kirschner, M. W. (1991). Cyclin is degraded by the ubiquitin pathway. Nature 349, 132–138.[CrossRef][Medline]
Gupta, G. P., and Massague, J. (2006). Cancer metastasis: building a framework. Cell 127, 679–695.[CrossRef][Medline]
Holland, A. J., and Taylor, S. S. (2006). Cyclin-B1-mediated inhibition of excess separase is required for timely chromosome disjunction. J. Cell. Sci 119, 3325–3336.
Liu, F. (2006). Smad3 phosphorylation by cyclin-dependent kinases. Cytokine Growth Factor Rev 17, 9–17.[CrossRef][Medline]
Liu, J. H., Wei, S., Burnette, P. K., Gamero, A. M., Hutton, M., and Djeu, J. Y. (1999). Functional association of TGF-beta receptor II with cyclin B. Oncogene 18, 269–275.[CrossRef][Medline]
Liu, W., Wu, G., Li, W., Lobur, D., and Wan, Y. (2007). Cdh1-APC targets Skp2 for destruction in TGF-{beta}-induced growth inhibition. Mol. Cell. Biol 27, 2967–2979.
Liu, X., Constantinescu, S. N., Sun, Y., Bogan, J. S., Hirsch, D., Weinberg, R. A., and Lodish, H. F. (2000). Generation of mammalian cells stably expressing multiple genes at predetermined levels. Anal. Biochem 280, 20–28.[CrossRef][Medline]
Massague, J. (2004). G1 cell-cycle control and cancer. Nature 432, 298–306.[CrossRef][Medline]
Massague, J., and Chen, Y. G. (2000). Controlling TGF-beta signaling. Genes Dev 14, 627–644.
Massague, J., and Wotton, D. (2000). Transcriptional control by the TGF-beta/Smad signaling system. EMBO J 19, 1745–1754.[CrossRef][Medline]
Orimo, A., Gupta, P. B., Sgroi, D. C., Arenzana-Seisdedos, F., Delaunay, T., Naeem, R., Carey, V. J., Richardson, A. L., and Weinberg, R. A. (2005). Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 121, 335–348.[CrossRef][Medline]
Palframan, W. J., Meehl, J. B., Jaspersen, S. L., Winey, M., and Murray, A. W. (2006). Anaphase inactivation of the spindle checkpoint. Science 313, 680–684.
Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R., Mosca, J. D., Moorman, M. A., Simonetti, D. W., Craig, S., and Marshak, D. R. (1999). Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147.
Reimann, J. D., Freed, E., Hsu, J. Y., Kramer, E. R., Peters, J. M., and Jackson, P. K. (2001). Emi1 is a mitotic regulator that interacts with Cdc20 and inhibits the anaphase promoting complex. Cell 105, 645–655.[CrossRef][Medline]
Roberts, A. B., Russo, A., Felici, A., and Flanders, K. C. (2003). Smad 3, a key player in pathogenetic mechanisms dependent on TGF-beta. Ann. NY Acad. Sci 995, 1–10.[Medline]
Roberts, A. B., Tian, F., Byfield, S. D., Stuelten, C., Ooshima, A., Saika, S., and Flanders, K. C. (2006). Smad3 is key to TGF-beta-mediated epithelial-to-mesenchymal transition, fibrosis, tumor suppression and metastasis. Cytokine Growth Factor Rev 17, 19–27.[CrossRef][Medline]
Siegel, P. M., and Massague, J. (2003). Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer. Nat. Rev. Cancer 3, 807–821.[CrossRef][Medline]
Stemmann, O., Zou, H., Gerber, S. A., Gygi, S. P., and Kirschner, M. W. (2001). Dual inhibition of sister chromatid separation at metaphase. Cell 107, 715–726.[CrossRef][Medline]
Tang, Z., Shu, H., Qi, W., Mahmood, N. A., Mumby, M. C., and Yu, H. (2006). PP2A is required for centromeric localization of Sgo1 and proper chromosome segregation. Dev. Cell 10, 575–585.[CrossRef][Medline]
Vodermaier, H. C. (2004). APC/C and SCF: controlling each other and the cell cycle. Curr. Biol 14, R787–R796.[CrossRef][Medline]
Waite, K. A., and Eng, C. (2003). From developmental disorder to heritable cancer: it's all in the BMP/TGF-beta family. Nat. Rev. Genet 4, 763–773.[CrossRef][Medline]
Wan, Y., Liu, X., and Kirschner, M. W. (2001). The anaphase-promoting complex mediates TGF-beta signaling by targeting SnoN for destruction. Mol. Cell 8, 1027–1039.[CrossRef][Medline]
Watanabe, M., and Whitman, M. (1999). The role of transcription factors involved in TGFbeta superfamily signaling during development. Cell Mol. Biol 45, 537–543.[Medline]
Wiseman, B. S., and Werb, Z. (2002). Stromal effects on mammary gland development and breast cancer. Science 296, 1046–1049.
Zhou, S., Lechpammer, S., Greenberger, J. S., and Glowacki, J. (2005). Hypoxia inhibition of adipocytogenesis in human bone marrow stromal cells requires transforming growth factor-beta/Smad3 signaling. J. Biol. Chem 280, 22688–22696.
This article has been cited by other articles:
|
|
 |
|
  A. Moustakas and C.-H. Heldin The regulation of TGF{beta} signal transduction Development, November 15, 2009; 136(22): 3699 - 3714. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
