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Msx homeobox genes inhibit differentiation through upregulation of cyclin D1

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, UMDNJ–Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA

View larger version (111K): [in a new window]   Fig. 1. Msx1 inhibits differentiation of multiple mesenchymal progenitor cell types. (A) C2C12 cells (a-d) or 10T1/2 cells (e-h) were infected with a control mammalian retrovirus (Vector) or retroviruses expressing Msx1 or Msx1A. Infected cells were grown under normal culture conditions (undifferentiated; a,e) or conditions that promote differentiation (differentiated; b-d,f-h) to myoctyes (b-d) or adipocytes (f-h). (B) Primary cultures of chicken limb chondrogenic cells (a-c) or calvarial osteogenic cells (e-g) were infected with a control avian retrovirus (Vector) or those expressing Msx1 or Msx1A. Differentiation is visualized by Alcian Blue staining (a-c) or silver staining (e-g). TMC23 chondrocytes (d) or BMP2T3 osteoblasts (h) were infected with a control mammalian retrovirus (Vector) or those expressing Msx1 or Msx1A followed by growth in conditions that promote differentiation. Alkaline phosphatase activity was measured from cell lysates; error bars show standard deviations of assays carried out in triplicate. (A,B) Representative data from experiments repeated a minimum of three times. Scale bars: 0.1 mm in A; 1 mm in B.

View larger version (44K): [in a new window]   Fig. 2. Msx1 does not promote cellular proliferation, but alters the expression of cell cycle regulators. (A) Proliferation assays were performed using chicken embryonic fibroblasts infected with a control retrovirus (Vector) or one expressing Msx1. Cells were plated at low density in media containing the indicated percentage of fetal bovine serum. Cell number was estimated by measuring the optical density of Napthal blue-black stained cells and is expressed as percent of the control; error bars indicate standard deviation of assays performed in triplicate. Shown are representative data from assays repeated a minimum of five times. (B) Northern blot analyses were performed using mRNA prepared from undifferentiated C2C12 cells that had been infected with a control retrovirus (Vector) or retroviruses expressing Msx1 or Msx1A. Northern blots contained 2.5 µg of mRNA per lane, and were probed with the indicated cDNAs and visualized by autoradiography. Northern blots were performed a minimum of two times using mRNA prepared from independent experiments; representative data are shown. RPL32 is a control for RNA loading.

View larger version (82K): [in a new window]   Fig. 3. Msx1 upregulates cyclin D1 expression and Cdk4 activity. (A) C2C12 cells were infected with a control retrovirus (Vector) or retroviruses expressing Msx1 or cyclin D1 and grown under normal culture conditions (a, undifferentiated) or conditions that promote differentiation (b-d, differentiated). Scale bar: 0.1 mm. (B,C) Western blot analysis was performed using extracts prepared from C2C12 cells infected with a control retrovirus (Vector) or with retroviruses expressing Msx1 or Msx1A, followed by cell growth under normal culture conditions (undifferentiated) or conditions that promote differentiation (differentiated). The panels show whole-cell extracts probed with the indicated antibodies, except for panels labeled -Cdk4 nuclear or -Cdk2 nuclear, in which nuclear extracts were used. RNA polymerase II (-RNA PolII) is a control for protein loading. (D) Cdk4 and Cdk2 kinase assays were performed using cell lysates from undifferentiated C2C12 cells or 293T cells infected with a control retrovirus (Vector) or those expressing Msx1 or Msx1A. The indicated kinases were immunoprecipitated using the corresponding antisera and kinase assays were performed using GST-Rb (amino acids 379-792) as substrate. Similar results were obtained using lysates prepared from differentiated C2C12 cells (data not shown).

View larger version (84K): [in a new window]   Fig. 4. Upregulation of cyclin D1 is a conserved activity of the Msx family. (A) C2C12 cells were infected with a control retrovirus (Vector) or retroviruses expressing the indicated homeoproteins and grown under conditions that promote differentiation. Scale bar: 0.1 mm. (B) Western blot analysis was performed using extracts prepared from C2C12 cells infected with a control retrovirus (Vector) or with retroviruses expressing the indicated homeoproteins, as in A. The panels show whole cell extracts probed with the indicated antibodies. Equivalent expression of the various homeoproteins is shown by -Myc, which detects the Myc epitope present at the N-terminal region of each homeoprotein. RNA polymerase II (-RNA PolII) is a control for protein loading.

View larger version (66K): [in a new window]   Fig. 5. Regions of Msx1 required for upregulation of cyclin D1 include its transcriptional domains. (A) Schematic diagrams showing the primary structures of Msx1 and Msx2; the percent identity in the N-terminal region, homeodomain and C-terminal regions are indicated. Also shown are the protein regions contained in the truncated Msx1 derivatives, Msx11, Msx12 and Msx13. (B) Western blot analyses were performed using extracts prepared from differentiated C2C12 cells following infection with a control retrovirus (Vector) or with retroviruses expressing the indicated Msx protein. Western blots were probed with -cyclin D1 or -MHC antibodies; -RNA PolII is a control for protein loading. (C) C2C12 cells were infected with a control retrovirus (Vector) or a retrovirus expressing an Msx1-estrogen receptor fusion protein (Msx11-ER). Infected cells were incubated in the presence (+) or absence (-) of tamoxifen (100 nM) and grown under conditions that promote differentiation. Scale bar: 0.1 mm. (D) Ribonuclease protection analyses were performed using total RNA prepared from C2C12 cells that had been infected with a control retrovirus (Vector) or with a retrovirus expressing Msx11-ER. Infected cells were treated with tamoxifen (100 nM) for the indicated times and ribonuclease protection assays were performed using a cyclin D1 riboprobe or an RPL32 riboprobe as a control for RNA loading.

View larger version (43K): [in a new window]   Fig. 6. Msx1 inhibits mammary epithelial differentiation and upregulates cyclin D1 in cell culture and in vivo. (A) Ribonuclease protection analyses were performed using total RNA obtained from HC11 mammary epithelial cells infected with a control retrovirus (Vector) or with those expressing Msx1 or Msx1A. Infected cells were grown under normal growth conditions (undifferentiated) or in conditions that promote differentiation (differentiated). (B) Ribonuclease protection analyses of total RNA prepared from the mammary fat pads of wild type (WT) and transgenic (TG) mice from virgin or pregnant females at the indicated stages. (A,B) Ribonuclease protection assays were performed using the indicated riboprobes; each lane contains 10 µg of total RNA, except for ß-casein in which 1.5 µg of RNA was used. RPL32 is a control for RNA loading.

View larger version (61K): [in a new window]   Fig. 7. Impaired mammary epithelial differentiation and increased cyclin D1 expression in MMTV-Msx1 transgenic mice. (A) Whole-mount Hematoxylin staining of mammary fat pads from female virgin, pregnant (14.5 dpc), or lactating MMTV-Msx1 transgenic mice and wild-type littermate controls, shown in low-power and high-power views. Although terminal end bud formation appears normal in the virgin transgenic and wild-type mice (arrows), retarded lobuloalveolar formation is observed in pregnant transgenics at 14.5 dpc (arrows). At this stage, and in lactating mice, there is reduced complexity of lobuloalveolar structures (arrows). (B) Immunohistochemical detection of cyclin D1 and Msx1 protein in sections from mammary glands of wild type and MMTV-Msx1 transgenic pregnant mice at 14.5 dpc. Note the elevated expression of Msx1 and cyclin D1 in nuclei of lumenal epithelial cells (arrows) in the mammary glands of the transgenic mice compared with their wild-type littermate controls. Scale bars: 0.5 mm in A; 0.05 mm in B.

View larger version (13K): [in a new window]   Fig. 8 A model for Msx function in cell cycle regulation. Msx1 indirectly upregulates cyclin D1 expression (red arrows), resulting in block of cell cycle exit (red bar) and subsequent differentiation. Although cyclin D1 can promote cellular proliferation (green arrow), Msx1 does not, presumably through its effects on other cell cycle components (broken red arrow). Thus, overexpression of Msx1 would block cell cycle exit without promoting proliferation, while loss of Msx1 activity might lead to premature cell cycle exit.

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