1 Center for Advanced Biotechnology and Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
2 Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
3 Department of Pediatrics, UMDNJRobert Wood Johnson Medical School, Piscataway, NJ 08854, USA
*Author for correspondence (e-mail: abate{at}cabm.rutgers.edu)
Accepted May 4, 2001
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SUMMARY |
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Key words: Cell cycle, Differentiation, Proliferation, Homeobox genes, Transgenic mice, Mammary gland
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INTRODUCTION |
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The vertebrate Msx homeobox gene family contains three members, two of which (Msx1 and Msx2) have been well studied with respect to their expression patterns and biochemical properties (reviewed by Bendall and Abate-Shen, 2000; Davidson, 1995). These genes encode closely related homeoproteins that function as transcriptional repressors through interactions with components of the core transcription complex as well as other homeoproteins (Catron et al., 1996; Catron et al., 1995; Zhang et al., 1996; Zhang et al., 1997). Both Msx1 and Msx2 are expressed in overlapping spatial and temporal domains during development, in discrete regions of the facial primordia, limbs, neural tube and other embryonic regions (Bendall and Abate-Shen, 2000; Davidson, 1995).
Although they are expressed in relatively diverse tissues, a common feature of Msx gene expression is its association with multipotent progenitor cells. For example, expression of Msx1 is robust in the progress zone of the limb bud, which corresponds to a region of highly proliferative, multipotential cells that give rise to chondrogenic and osteogenic derivatives of the limb. In contrast, Msx1 is not expressed in the more proximal regions of the limb, where cells have ceased to proliferate and have begun to undergo differentiation (Bendall and Abate-Shen, 2000; Davidson, 1995). These, and other observations, have led to the hypothesis that Msx genes promote cellular proliferation, and that their expression is inversely correlated with differentiation (Bendall et al., 1999; Dodig et al., 1999; Mina et al., 1996; Pavlova et al., 1994; Song et al., 1992; Woloshin et al., 1995).
This model has garnered support from cell culture data, which have shown that forced expression of Msx1 in myogenic precursors blocks their differentiation and represses expression of lineage specific genes, such as MyoD (Myod1 Mouse Genome Informatics) (Song et al., 1992; Woloshin et al., 1995). Moreover, ectopic expression of Msx1 during chicken embryogenesis inhibits development of the limb musculature and represses MyoD expression in vivo (Bendall et al., 1999). Msx genes have also been implicated as inhibitors of chondrogenic and osteogenic differentiation in culture (Dodig et al., 1999; Mina et al., 1996), suggesting that these activities may not be limited to the myogenic lineage. Furthermore, the phenotypes of Msx loss- or gain-of-function mutations are also consistent with roles for Msx genes in regulating cellular proliferation and differentiation in vivo (Jabs et al., 1993; Liu et al., 1995; Satokata et al., 2000; Satokata and Maas, 1994; Wilkie et al., 2000; Winograd et al., 1997).
We have explored the mechanism(s) by which Msx genes, particularly Msx1, function as negative regulators of differentiation. Using both cell culture and in vivo model systems, we demonstrate that Msx1 inhibits the differentiation of multiple mesenchymal and epithelial cell types. This inhibition is associated with upregulation of cyclin D1 (Ccnd1 Mouse genome Informatics) expression as well as Cdk4 activity, while Msx1 has minimal effects on cellular proliferation. Transgenic mice that overexpress Msx1 in the mammary gland display significant defects in epithelial differentiation during pregnancy, which are correlated with increased cyclin D1 expression. We propose a model in which Msx1 prevents exit from the cell cycle by maintaining high levels of cyclin D1 expression, thereby blocking terminal differentiation of progenitor cell populations.
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MATERIALS AND METHODS |
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C2C12 cells were grown in Dubleccos Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum (FBS). Myogenic differentiation was initiated by incubating confluent cells for 3 days in DMEM containing 2% horse serum and verified by visual detection of myotubes and by western blot analysis using an MHC antibody (see below). 10T1/2 cells (ATCC) were grown in Basal Medium Eagle (BME) supplemented with 15% FBS. Adipocyte differentiation was initiated by treatment with 3 µM 5-azacytidine for 24 hours. After retroviral infection, confluent cells were treated with 1 µM dexamethasone for 7 days.
Micromass assays were performed essentially as described (Ahrens et al., 1977). White Leghorn chicken eggs (SPAFAS) were incubated to stage 21-22 (Hamburger and Hamilton, 1951). Forelimb buds were collected and digested with 0.1% trypsin-0.1% collagenase. 10 µl of digested cells (2x107 cells/ml) were infected with 1 µl of concentrated avian retroviruses (109 cfu/ml) in growth media (F12/DMEM supplemented with 10% FBS) containing 8 µg/ml polybrene. Infected cells were incubated for 4 days and differentiation was verified by staining with 0.5% Alcian Blue 8GX (pH 1). TMC23 cells (Xu et al., 1998) were grown in DMEM supplemented with 10% FBS. Differentiation was initiated by treating confluent cells were with 50 µg/ml ascorbic acid and 4 mM ß-glycerol phosphate for 16 days.
Chicken calvarial osteoblasts were prepared essentially as described (Gerstenfeld et al., 1987). Eggs were incubated to stage 42 (Hamburger and Hamilton, 1951) after which the calvaria (skull bone) were digested with 0.25% trypsin-0.2% collagenase. Digested cells were plated at 5000 cells/cm2 in -Minimum Eagle media (
-MEM) supplemented with 10% FBS. Differentiation was initiated by addition of 100 µg/ml ascorbic acid and 5 mM ß-glycerol phosphate for 16 days and verified by staining with Von Kossas stain (silver staining). BMP2T3 calvarial cells (Ghosh-Choudhury et al., 1996) were grown in
MEM supplemented with 7% FBS. Differentiation was initiated by treating confluent cells with 100 µg/ml ascorbic acid and 5 mM ß-glycerol phosphate for 16 days.
HC11 mammary epithelial cells (Hynes et al., 1990) were grown in RPMI 1640 media supplemented with 10% FBS, 5 µg/ml insulin and 10 ng/ml epidermal growth factor (EGF). Differentiation was initiated by removal of EGF for 3 days, followed by treatment with 10 µM dexamethasone, 5 µg/ml prolactin and 5 µg/ml insulin.
Expression analysis and probes
Northern blot analysis was performed using mRNA (2.5 µg per lane) prepared by the Poly(A)Pure mRNA purification kit (Ambion). Probes used for northern Blot analysis were labeled by StripEZ DNA labeling kit (Ambion) and are described in the text. Ribonuclease protection assays were performed as described (Shen and Leder, 1992) using total RNA prepared with Trizol RNA Isolation Reagent (GIBCO-BRL). The Msx1 riboprobe corresponds to sequences encoding amino acids 1-57. The riboprobe for ß-casein corresponds to sequences encoding amino acids 126 to 134. The rpL32 riboprobe has been described (Shen and Leder, 1992). Northern blot and ribonuclease protection assays were quantitated using a phosphorimager (Molecular Dynamics).
Western blot analysis, antibodies and kinase assays
Western blot analysis was performed with an ECL-Plus chemiluminescence kit (Amersham Pharmacia Biotech) using the following antibodies: monoclonal antibody against myosin heavy chain (MF20) from Developmental Studies Hybridoma Bank; monoclonal antibody against murine cyclin D1 from Oncogene Sciences; polyclonal antisera against Cdk4 and Cdk2 from Santa Cruz; and polyclonal antisera against RNA polymerase II small subunit band 7, a gift from Danny Reinberg. The monoclonal antibody directed against murine Msx1 was generated using the bacterially expressed full-length Msx1 protein as antigen. This antibody specifically recognizes an N-terminal epitope of the mouse Msx1 protein and does not crossreact with Msx2 (G. H. and C. A.-S., unpublished). Cdk4 and Cdk2 kinase assays were performed as described (Parry et al., 1999). Cell lysates (500 µg) were immunoprecipitated with 10 µl of anti-Cdk4 or anti-Cdk2 antibody plus 10 µl of protein-A/G agarose beads (Santa Cruz). Kinase assays were performed using GST-RB (amino acids 379-792, 0.5 µg) as substrate.
Generation and analysis of MMTV-Msx1 transgenes
Transgenic mice were generated as described (Stewart et al., 1984) in FVB/N (Taconic Biotechnology) mice. Founders were identified by Southern blot analysis, using genomic DNA digested with BamHI and an Msx1 cDNA probe; progeny were genotyped by PCR. Three independent transgenic lines were established; two lines expressed the Msx1 transgene specifically in the mammary gland. Results shown are from one of these lines; the other line had a similar phenotype. No additional mammary epithelial abnormalities have been detected during 1 year of observation (data not shown).
For whole-mount and immunohistochemical analyses, mammary fat pads from virgin or pregnant transgenic female mice or littermate controls were dissected, spread onto glass slides, and fixed in 10% formalin; in each case the posterior fat pad (#4) was examined. For whole mounts, the mammary pads were stained with Hematoxylin. For immunohistochemistry, the formalin-fixed mammary pads were embedded in paraffin and adjacent sections were stained with either anti-Msx1 or anti-cyclin D1 antibodies (as above). Staining was performed using Vector M.O.M. Immunodection kit and Vector NovaRED substrate kit (Vector Laboratories). For ribonuclease protection analysis, total RNA was prepared (as above) from the anterior fat pad (#3).
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RESULTS |
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We observed a similar ability of Msx1 to inhibit differentiation of adipocytes, chondrocytes and osteoblasts. In particular, misexpression of Msx1 in 10T1/2 fibroblasts, which have the potential to undergo differentiation into multiple cell types, including adipocytes (Reznikoff et al., 1973), resulted in a failure of the 10T1/2 cells to undergo adipogenesis, whereas overexpression of Msx1A had no significant effect (Fig. 1A, e-h). In micromass explant assays, in which mesenchymal cells from chicken forelimb buds form chondrogenic nodules in culture (Ahrens et al., 1977), misexpression of Msx1, but not of Msx1A, significantly inhibited chondrogenesis as evident by reduced Alcian Blue staining (Fig. 1B, a-c). Similarly, misexpression of Msx1, but not of Msx1A, resulted in a sevenfold inhibition of chondrocyte differentiation in a murine chondrocyte cell line, TMC23 (Xu et al., 1998) (Fig. 1B, d). Finally, Msx1, but not Msx1A, inhibited osteogenesis in chicken calvarial osteoblasts (Fig. 1B, e-g) as well as in a murine calvarial cell line, BMP2T3 (Ghosh-Choudhury et al., 1996) (Fig. 1B, h). In summary, we have found that Msx1 expression is incompatible with differentiation of multiple mesenchymal lineages, including muscle, fat, cartilage and bone, which confirms and extend previous findings (Bendall et al., 1999; Dodig et al., 1999; Mina et al., 1996; Song et al., 1992; Woloshin et al., 1995).
In contrast to its potent effect on differentiation, misexpression of Msx1 had a minimal effect (10-20% activation) on cellular proliferation of embryonic fibroblasts or various other cell types (Fig. 2A; data not shown). This observation was unexpected as Msx genes have been thought to promote cellular proliferation, based on their expression in proliferating cells in vivo (reviewed in Bendall et al., 1999). Taken together, we conclude that Msx1 is a potent inhibitor of cellular differentiation for multiple mesenchymal lineages, but does not significantly promote cellular proliferation.
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Of the cell cycle regulatory genes examined, we found that the most prominent effect of Msx1 misexpression was a robust increase in cyclin D1 expression (20- to 30-fold), whereas misexpression of the Msx1A mutant had no significant effect (Fig. 2B). In contrast, misexpression of Msx1 modestly repressed cyclin D3, cyclin E, cyclin B1, Rb1 and p21WAF1/CIP, while it had little or no effect on cyclin D2, cyclin A2, p27KIP1, p57KIP2 and p16INK4a. The finding that Msx1 upregulates cyclin D1 but not cyclin E or the G2 cyclins (cyclin A2 and cyclin B1), is consistent with our observation that Msx1 inhibits differentiation without promoting proliferation. Moreover, we have observed that a greater percentage of Msx1-expressing cells are found in the G1 phase by flow cytometry (G. H. and C. A.-S., unpublished). A similar upregulation of cyclin D1 after misexpression of Msx1 was observed in the adipocyte, chondrocyte and osteoblast lineages (data not shown).
cyclin D1 misexpression has been shown to inhibit differentiation of myogenic cells in culture (Skapek et al., 1995) and is therefore an excellent candidate for a primary downstream mediator of Msx1 activity. To assess whether the effect of misexpressing cyclin D1 is analogous to that of Msx1, we examined myogenic differentiation following misexpression of either gene in C2C12 cells (Fig. 3A). Similar to Msx1, misexpression of cyclin D1 resulted in marked inhibition of myogenic differentiation, apparent from the absence of myotube formation. Moreover, in addition to upregulating cyclin D1 mRNA, Msx1 upregulated cyclin D1 protein, which was inversely correlated with inhibition of MHC expression (Fig. 3B). This result underscores the relationship between inhibition of differentiation by Msx1 and upregulation of cyclin D1.
The cell cycle regulatory activities of cyclin D1 are mediated through its interaction with catalytic partners, particularly Cdk4 (Sherr and Roberts, 1999). Therefore, we asked whether Msx1 misexpression altered the protein level or activity of Cdk4. While Msx1 misexpression did not alter the overall levels of total cellular Cdk4, the accumulation of Cdk4 in the nucleus was significantly increased relative to controls (Fig. 3C). Co-immunoprecipitation analysis with anti-cyclin D1 antibody confirmed an increased association of Cdk4 with cyclin D1 in Msx1-expressing cells (data not shown). Moreover, Cdk4 kinase activity was also substantially elevated in Msx1-expressing cells, including C2C12 cells and a human kidney cell line, 293T (Fig. 3D). In contrast, misexpression of Msx1 had no significant effect on the nuclear accumulation (Fig. 3C) or kinase activity (Fig. 3D) of Cdk2, the major partner of cyclin E. These findings are consistent with the observation that Msx1 inhibits differentiation but does not promote cellular proliferation. Taken together, these data suggest that Msx1 inhibits cellular differentiation through upregulation of cyclin D1 and activation of Cdk4 activity.
Upregulation of cyclin D1 is a conserved activity specific for the Msx family
The Msx2 homeobox gene has many similarities to Msx1 with respect to its structure, expression pattern, biochemical properties, and biological functions (Bendall and Abate-Shen, 2000; Catron et al., 1996; Davidson, 1995; Satokata et al., 2000). We have found that Msx2 also inhibits differentiation of multiple mesenchymal lineages (Fig. 4A and data not shown), and therefore we examined whether its misexpression also affected cyclin D1 expression. Indeed, we found that misexpression of Msx2 upregulated cyclin D1 to a similar extent as Msx1, and was inversely correlated with inhibition of MHC expression and myotube formation (Figs 4A,B, 5B). In contrast, two members of the Dlx homeobox gene family (Dlx2 and Dlx5), which are closely related to the Msx family (Bendall and Abate-Shen, 2000), inhibited differentiation but did not upregulate cyclin D1 (Fig. 4A,B). Furthermore, two members of the Hox gene family (Hoxa7 and Hoxc8), which are less closely related to the Msx family did not inhibit differentiation or upregulate cyclin D1 (Fig. 4A,B). Therefore, we conclude that inhibition of differentiation through upregulation of cyclin D1 is a conserved activity that is specific for the Msx family.
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Next, we generated a chimeric fusion protein in which the essential regions of Msx1, namely the N-terminal region and the homeodomain, were fused with the hormone-binding domain of a tamoxifen-responsive mutant of the estrogen receptor (Littlewood et al., 1995). Infection of C2C12 cells with a retrovirus expressing this Msx11-ER chimeric gene resulted in tamoxifen-dependent inhibition of differentiation (Fig. 5C) that was comparable with that of native Msx1 (see Fig. 1A). We used this estrogen receptor-regulated expression system to evaluate the time course for activation of cyclin D1 by Msx1. In Msx1
1-ER-infected cells, cyclin D1 expression increased as early as 2 hours after addition of tamoxifen and peaked by 24 hours (Fig. 5D), suggesting that cyclin D1 represents an early response gene for Msx1. However, we were unable to address whether cyclin D1 is a direct target of Msx1 using cycloheximide treatment, as addition of cycloheximide by itself increased cyclin D1 mRNA levels (data not shown).
The ability for Msx1 to inhibit differentiation and upregulate cyclin D1 primarily requires the N-terminal domain (Fig. 5A,B); notably, this region contains the major regulatory domains defined in transcription assays (Catron et al., 1996; Catron et al., 1995). However, as Msx proteins are potent transcriptional repressors and have no reported activator potential (Catron et al., 1995), it is likely that Msx1 upregulates cyclin D1 indirectly rather than by direct activation through cyclin D1 promoter elements. Indeed, we have found that Msx1 does not activate the cyclin D1 promoter in transient transfection assays, irrespective of the cell type or the human or rat cyclin D1 promoter/enhancer regions examined (G. H. and C. A.-S., unpublished).
Msx1 inhibits mammary epithelial differentiation and upregulates cyclin D1 in transgenic mice
To extend our findings, we developed a transgenic mouse system to examine the consequences of Msx1 misexpression for differentiation and cyclin D1 expression in vivo. We chose to investigate the mammary gland because of the known expression and functional significance of Msx and cyclin D1 genes in this tissue. In particular, both Msx1 and Msx2 are expressed in the mammary epithelium during embryogenesis and pregnancy, and loss of Msx gene function results in profound defects in mammary gland morphogenesis during development (Friedmann and Daniel, 1996; Phippard et al., 1996; Satokata et al., 2000). Moreover, cyclin D1 is essential for mammary gland differentiation and function during pregnancy (Fantl et al., 1999; Fantl et al., 1995; Sicinski et al., 1995).
Initially, we asked whether Msx1 also inhibited differentiation of mammary epithelial cells in culture, analogous to its effects on mesenchymal cell types (Fig. 6A). For this purpose, we used HC11 cells, which undergo terminal differentiation and produce milk proteins, including ß-casein, after treatment with lactogenic hormones (Hynes et al., 1990). We found that differentiation of HC11 cells was inhibited by misexpression of Msx1, but not by Msx1A, as determined by the nearly complete absence of ß-casein (Csnb Mouse Genome Informatics) expression, and that this inhibition correlated with upregulation of cyclin D1 expression (Fig. 6A).
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DISCUSSION |
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In addition to the Msx gene family, numerous other homeobox genes have been implicated in controlling aspects of cellular proliferation and differentiation during embryogenesis, while their aberrant expression is associated with cellular transformation. The most well studied in this regard are the Hox genes, which are the largest family of homeobox genes (Krumlauf, 1994). Although loss-of-function mutation of members of the Hox family produces distinct phenotypic outcomes, certain common themes also emerge. Notably, Hox mutants often display vertebral transformations that have been interpreted as changes in rates of cell proliferation and/or survival (Duboule, 1995). Hox genes, as well as their essential co-factors, the Pbx genes, are aberrantly expressed in leukemias and many solid tumors, and their forced expression is associated with cellular transformation (Cillo et al., 1999; Maulbecker and Gruss, 1993). Furthermore, the Gax homeobox gene negatively regulates cardiomyocyte proliferation through upregulation of the cell cycle regulatory gene, p21WAF1/CIP (Smith et al., 1997). Thus, the link between homeobox gene function and regulation of the cell cycle machinery is likely to emerge as a prevalent theme.
cyclin D1 is a downstream effector of Msx gene function
cyclin D1 inhibits differentiation of multiple lineages through its ability to block exit from the cell cycle, and thus represents an excellent candidate downstream effector for Msx1 in culture (Sherr and Roberts, 1999; Walsh and Perlman, 1997). Indeed, cyclin D1 blocks MyoD activity in myogenic progenitors (Rao and Kohtz, 1995; Skapek et al., 1995), which is noteworthy as Msx1 inhibits MyoD expression in cell culture as well as in vivo (Bendall et al., 1999; Woloshin et al., 1995). However, the consequences of Msx1 misexpression are not entirely analogous to those of cyclin D1, as Msx1 does not significantly promote cellular proliferation, resulting in a greater percentage of Msx1-expressing cells in the G1 phase. This distinction probably reflects the fact that Msx1 upregulates cyclin D1 but not cyclin E or the G2 cyclins.
In addition, cyclin D1 is an excellent candidate downstream effector of Msx in vivo. Indeed, we have demonstrated that overexpression of Msx1 in the mammary gland results in upregulation of cyclin D1 expression. Furthermore, the expression patterns of Msx1 and Msx2 overlap with that of cyclin D1 at early stages of embryogenesis in the primitive streak and lateral mesoderm and later in the limb mesenchyme, neural tube, craniofacial mesenchyme, retina and mammary gland (Wianny et al., 1998; G. H. and C. A.-S., unpublished). Moreover, the phenotypes of Msx1, Msx2 and cyclin D1 loss-of-function mutations share similarities, but are not identical, as discussed below. Our current findings do not preclude negative regulation by Msx genes of lineage-specific genes such as MyoD or osteocalcin, which are involved in terminal differentiation (Bendall et al., 1999; Newberry et al., 1997; Woloshin et al., 1995), but instead expand the repertoire of their downstream effectors to include global cell cycle regulators.
Although we have found that cyclin D1 is activated as an early response gene of Msx1, the available evidence suggests that it is likely to be an indirect target. Indeed, Msx genes encode transcriptional repressors and have not been previously implicated as transcriptional activators (Catron et al., 1996; Catron et al., 1995). Given the known significance of protein-protein interactions for mediating Msx function (Bendall et al., 1999; Zhang et al., 1997), it seems plausible that Msx1 activates cyclin D1 through interaction with other transcriptional regulators, perhaps those directly upstream of cyclin D1.
A model for Msx gene function
Based on our findings, we propose a model for the mechanism of Msx gene function in cellular proliferation and differentiation (Fig. 8). We find that Msx genes maintain cells in a proliferative state by blocking exit from the cell cycle, while not actively promoting proliferation. Moreover, this model explains the apparent activity of Msx genes in inhibiting cellular differentiation as a consequence of preventing cell cycle exit. Thus, the model distinguishes between a direct effect on the cell cycle and an indirect effect on differentiation.
Our model provides a basis for reconciling the observed similarities between the phenotypes of Msx loss-of-function and gain-of-function mutations (Jabs et al., 1993; Liu et al., 1995; Satokata et al., 2000; Satokata and Maas, 1994; Wilkie et al., 2000; Winograd et al., 1997). For example, mutant mice lacking either Msx2 or both Msx1 and Msx2 display profound defects in mammary gland development that have been interpreted as arrested differentiation (Satokata et al., 2000). In the context of our model, loss of Msx function is predicted to result in premature exit of progenitor cells from the cell cycle, resulting in decreased proliferation and consequently impaired morphogenesis. Conversely, in MMTV-Msx1 transgenic mice, which also display defects in mammary gland differentiation, overexpression of Msx genes is predicted to block progenitor cells from exiting the cell cycle; in the absence of increased proliferation, the outcome would also be impaired morphogenesis. Comparable arguments can be made for the similar phenotypes found in loss- and gain-of-function analyses of Msx function in cranial development (Jabs et al., 1993; Liu et al., 1995; Satokata et al., 2000; Satokata and Maas, 1994; Wilkie et al., 2000; Winograd et al., 1997). In addition, similar phenotypes following both loss- and gain-of-function mutations have been reported for other homeobox genes (e.g. Pollock et al., 1992).
cyclin D1 loss-of-function mutant mice display delayed maturation of the mammary gland that has been interpreted as partial blockage of differentiation (Fantl et al., 1999; Fantl et al., 1995; Sicinski et al., 1995). In particular, these mice undergo normal early proliferation and side-branching, but greatly reduced lobuloalveolar formation (Fantl et al., 1999), resembling the phenotype of MMTV-Msx1 transgenic mice. In cyclin D1 loss-of-function mutants, progenitor cells would be predicted to exit the cell cycle prematurely, resulting in less proliferation and hence impaired morphogenesis; counterintuitively, the outcome would thus be similar to that of MMTV-Msx1 transgenic mice. Conversely, MMTV-cyclin D1 transgenic mice display mammary epithelial hyperproliferation that ultimately leads to carcinoma (Wang et al., 1994), consistent with the well-known activity of cyclin D1 in promoting cellular proliferation, unlike Msx1.
The potential for Msx genes to upregulate cyclin D1 has important implications for mammary carcinogenesis. Upregulation of cyclin D1 protein expression is one of the most prevalent alterations in breast carcinoma, occurring in approximately 40% of cases (Hall and Peters, 1996). However, cyclin D1 gene amplification occurs in only a fraction of these cases, indicating that other factors are responsible for upregulating cyclin D1. Thus, our findings suggest that a potential role for Msx genes in breast carcinogenesis deserves further study.
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ACKNOWLEDGMENTS |
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