Epimorphin mediates mammary luminal morphogenesis through control of C/EBPbeta - PubMed (original) (raw)
Epimorphin mediates mammary luminal morphogenesis through control of C/EBPbeta
Y Hirai et al. J Cell Biol. 2001.
Abstract
We have shown previously that epimorphin (EPM), a protein expressed on the surface of myoepithelial and fibroblast cells of the mammary gland, acts as a multifunctional morphogen of mammary epithelial cells. Here, we present the molecular mechanism by which EPM mediates luminal morphogenesis. Treatment of cells with EPM to induce lumen formation greatly increases the overall expression of transcription factor CCAAT/enhancer binding protein (C/EBP)beta and alters the relative expression of its two principal isoforms, LIP and LAP. These alterations were shown to be essential for the morphogenetic activities, since constitutive expression of LIP was sufficient to produce lumen formation, whereas constitutive expression of LAP blocked EPM-mediated luminal morphogenesis. Furthermore, in a transgenic mouse model in which EPM expression was expressed in an apolar fashion on the surface of mammary epithelial cells, we found increased expression of C/EBPbeta, increased relative expression of LIP to LAP, and enlarged ductal lumina. Together, our studies demonstrate a role for EPM in luminal morphogenesis through control of C/EBPbeta expression.
Figures
Figure 1
Effects of apolar presentation of EPM. (A) EPM activates luminal morphogenesis. PTSE cell clusters were cultured in collagen for 4 d in the absence of tetracycline (EPM ON) (a), then cultured for an additional 4 d in the presence of tetracycline (EPM OFF) and with (b) or without (c) H12 form of EPM in the medium. The luminal diameter of >20 clusters in each category was measured; graph bar indicates SD. (B) EPM increases C/EBPβ expression and the LIP/LAP ratio. PTSE cell clusters were cultured on plastic in the presence or absence of tetracycline, and SCp2 cells were cultured on recombinant full-length EPM (rEPM) or collagen (Hirai et al. 1998) or in the presence of rEPM/H12. Analyses of unclustered SCp2 cells incubated on tissue culture plastic or plastic coated with collagen are shown as controls. CRM, cross-reactive material (Seagroves et al. 1998). Estimated LIP/LAP ratios relative to the control samples are indicated. (C) LIP and LAP are dramatically upregulated by both EPM transfection (sig-EPM) and addition of rEPM to primary mammary epithelial cells. The identity of the band between CRM and LAP is unknown. (D) SCp2 cells cultured in the presence of rEPM also upregulate C/EBPα. For B, C, and D, the results are typical of three independent experiments. Bar, 100 μm.
Figure 1
Effects of apolar presentation of EPM. (A) EPM activates luminal morphogenesis. PTSE cell clusters were cultured in collagen for 4 d in the absence of tetracycline (EPM ON) (a), then cultured for an additional 4 d in the presence of tetracycline (EPM OFF) and with (b) or without (c) H12 form of EPM in the medium. The luminal diameter of >20 clusters in each category was measured; graph bar indicates SD. (B) EPM increases C/EBPβ expression and the LIP/LAP ratio. PTSE cell clusters were cultured on plastic in the presence or absence of tetracycline, and SCp2 cells were cultured on recombinant full-length EPM (rEPM) or collagen (Hirai et al. 1998) or in the presence of rEPM/H12. Analyses of unclustered SCp2 cells incubated on tissue culture plastic or plastic coated with collagen are shown as controls. CRM, cross-reactive material (Seagroves et al. 1998). Estimated LIP/LAP ratios relative to the control samples are indicated. (C) LIP and LAP are dramatically upregulated by both EPM transfection (sig-EPM) and addition of rEPM to primary mammary epithelial cells. The identity of the band between CRM and LAP is unknown. (D) SCp2 cells cultured in the presence of rEPM also upregulate C/EBPα. For B, C, and D, the results are typical of three independent experiments. Bar, 100 μm.
Figure 1
Effects of apolar presentation of EPM. (A) EPM activates luminal morphogenesis. PTSE cell clusters were cultured in collagen for 4 d in the absence of tetracycline (EPM ON) (a), then cultured for an additional 4 d in the presence of tetracycline (EPM OFF) and with (b) or without (c) H12 form of EPM in the medium. The luminal diameter of >20 clusters in each category was measured; graph bar indicates SD. (B) EPM increases C/EBPβ expression and the LIP/LAP ratio. PTSE cell clusters were cultured on plastic in the presence or absence of tetracycline, and SCp2 cells were cultured on recombinant full-length EPM (rEPM) or collagen (Hirai et al. 1998) or in the presence of rEPM/H12. Analyses of unclustered SCp2 cells incubated on tissue culture plastic or plastic coated with collagen are shown as controls. CRM, cross-reactive material (Seagroves et al. 1998). Estimated LIP/LAP ratios relative to the control samples are indicated. (C) LIP and LAP are dramatically upregulated by both EPM transfection (sig-EPM) and addition of rEPM to primary mammary epithelial cells. The identity of the band between CRM and LAP is unknown. (D) SCp2 cells cultured in the presence of rEPM also upregulate C/EBPα. For B, C, and D, the results are typical of three independent experiments. Bar, 100 μm.
Figure 1
Effects of apolar presentation of EPM. (A) EPM activates luminal morphogenesis. PTSE cell clusters were cultured in collagen for 4 d in the absence of tetracycline (EPM ON) (a), then cultured for an additional 4 d in the presence of tetracycline (EPM OFF) and with (b) or without (c) H12 form of EPM in the medium. The luminal diameter of >20 clusters in each category was measured; graph bar indicates SD. (B) EPM increases C/EBPβ expression and the LIP/LAP ratio. PTSE cell clusters were cultured on plastic in the presence or absence of tetracycline, and SCp2 cells were cultured on recombinant full-length EPM (rEPM) or collagen (Hirai et al. 1998) or in the presence of rEPM/H12. Analyses of unclustered SCp2 cells incubated on tissue culture plastic or plastic coated with collagen are shown as controls. CRM, cross-reactive material (Seagroves et al. 1998). Estimated LIP/LAP ratios relative to the control samples are indicated. (C) LIP and LAP are dramatically upregulated by both EPM transfection (sig-EPM) and addition of rEPM to primary mammary epithelial cells. The identity of the band between CRM and LAP is unknown. (D) SCp2 cells cultured in the presence of rEPM also upregulate C/EBPα. For B, C, and D, the results are typical of three independent experiments. Bar, 100 μm.
Figure 2
Minimal proteolysis of LAP occurs during sample preparation. (A) Diagram depicting the targeted location of the anti-LAPonly antibody and of the commercial anti-C/EBPβ antibody. (B) Western blot probed with the anti-LAPonly antibody. (C) Western blots probed with commercial anti-C/EBPβ antibody. Left: Parallel blot to B; asterisk, cross-reactive material. Right: Blot of SCp2 cells transiently transfected with LAP or LIP expression plasmids. Results shown are typical of two independent experiments.
Figure 2
Minimal proteolysis of LAP occurs during sample preparation. (A) Diagram depicting the targeted location of the anti-LAPonly antibody and of the commercial anti-C/EBPβ antibody. (B) Western blot probed with the anti-LAPonly antibody. (C) Western blots probed with commercial anti-C/EBPβ antibody. Left: Parallel blot to B; asterisk, cross-reactive material. Right: Blot of SCp2 cells transiently transfected with LAP or LIP expression plasmids. Results shown are typical of two independent experiments.
Figure 2
Minimal proteolysis of LAP occurs during sample preparation. (A) Diagram depicting the targeted location of the anti-LAPonly antibody and of the commercial anti-C/EBPβ antibody. (B) Western blot probed with the anti-LAPonly antibody. (C) Western blots probed with commercial anti-C/EBPβ antibody. Left: Parallel blot to B; asterisk, cross-reactive material. Right: Blot of SCp2 cells transiently transfected with LAP or LIP expression plasmids. Results shown are typical of two independent experiments.
Figure 3
Characterization of clones that conditionally express LIP and LAP. (A) Analysis of C/EBPβ gene products in LIP and LAP transfectants cultured in the presence (5 μg/ml) or absence of tetracycline (tet). Results shown are typical of three independent experiments. (B and C) Behavior of LIP-transfected cells cultured in various concentrations of tetracycline. Clusters of SCp2 controls (B, a) and of SCp2/LIP1 and SCp2/LIP2 in 0.5 μg/ml tet (B, b) formed compact colonies in collagen (shown in C, a for SCp2/LIP1; 0.5 μg/ml tetracycline). SCp2/LIP3 expresses moderate levels of LIP and forms lumina in the presence of tetracycline (B c and C b). Reduction of tetracycline in medium of SCp2/LIP1 and SCp2/LIP2 cells results in apoptotic cell death (B, b; shown in C, c for SCp2/LIP1; 0.02 μg/ml tetracycline). (D) Induction of LAP transgene inhibits luminal morphogenesis. (a) LAP expression in clustered parental SCp2 cells. (b and c) Clustered parental Scp2 cells and SCp2/LAP cells cultured in collagen gels in the absence of tetracycline (tet) and in the presence and absence of rEPM. For B, C, and D, ≥20 colonies from each condition were examined. Bar, 100 μm.
Figure 3
Characterization of clones that conditionally express LIP and LAP. (A) Analysis of C/EBPβ gene products in LIP and LAP transfectants cultured in the presence (5 μg/ml) or absence of tetracycline (tet). Results shown are typical of three independent experiments. (B and C) Behavior of LIP-transfected cells cultured in various concentrations of tetracycline. Clusters of SCp2 controls (B, a) and of SCp2/LIP1 and SCp2/LIP2 in 0.5 μg/ml tet (B, b) formed compact colonies in collagen (shown in C, a for SCp2/LIP1; 0.5 μg/ml tetracycline). SCp2/LIP3 expresses moderate levels of LIP and forms lumina in the presence of tetracycline (B c and C b). Reduction of tetracycline in medium of SCp2/LIP1 and SCp2/LIP2 cells results in apoptotic cell death (B, b; shown in C, c for SCp2/LIP1; 0.02 μg/ml tetracycline). (D) Induction of LAP transgene inhibits luminal morphogenesis. (a) LAP expression in clustered parental SCp2 cells. (b and c) Clustered parental Scp2 cells and SCp2/LAP cells cultured in collagen gels in the absence of tetracycline (tet) and in the presence and absence of rEPM. For B, C, and D, ≥20 colonies from each condition were examined. Bar, 100 μm.
Figure 3
Characterization of clones that conditionally express LIP and LAP. (A) Analysis of C/EBPβ gene products in LIP and LAP transfectants cultured in the presence (5 μg/ml) or absence of tetracycline (tet). Results shown are typical of three independent experiments. (B and C) Behavior of LIP-transfected cells cultured in various concentrations of tetracycline. Clusters of SCp2 controls (B, a) and of SCp2/LIP1 and SCp2/LIP2 in 0.5 μg/ml tet (B, b) formed compact colonies in collagen (shown in C, a for SCp2/LIP1; 0.5 μg/ml tetracycline). SCp2/LIP3 expresses moderate levels of LIP and forms lumina in the presence of tetracycline (B c and C b). Reduction of tetracycline in medium of SCp2/LIP1 and SCp2/LIP2 cells results in apoptotic cell death (B, b; shown in C, c for SCp2/LIP1; 0.02 μg/ml tetracycline). (D) Induction of LAP transgene inhibits luminal morphogenesis. (a) LAP expression in clustered parental SCp2 cells. (b and c) Clustered parental Scp2 cells and SCp2/LAP cells cultured in collagen gels in the absence of tetracycline (tet) and in the presence and absence of rEPM. For B, C, and D, ≥20 colonies from each condition were examined. Bar, 100 μm.
Figure 3
Characterization of clones that conditionally express LIP and LAP. (A) Analysis of C/EBPβ gene products in LIP and LAP transfectants cultured in the presence (5 μg/ml) or absence of tetracycline (tet). Results shown are typical of three independent experiments. (B and C) Behavior of LIP-transfected cells cultured in various concentrations of tetracycline. Clusters of SCp2 controls (B, a) and of SCp2/LIP1 and SCp2/LIP2 in 0.5 μg/ml tet (B, b) formed compact colonies in collagen (shown in C, a for SCp2/LIP1; 0.5 μg/ml tetracycline). SCp2/LIP3 expresses moderate levels of LIP and forms lumina in the presence of tetracycline (B c and C b). Reduction of tetracycline in medium of SCp2/LIP1 and SCp2/LIP2 cells results in apoptotic cell death (B, b; shown in C, c for SCp2/LIP1; 0.02 μg/ml tetracycline). (D) Induction of LAP transgene inhibits luminal morphogenesis. (a) LAP expression in clustered parental SCp2 cells. (b and c) Clustered parental Scp2 cells and SCp2/LAP cells cultured in collagen gels in the absence of tetracycline (tet) and in the presence and absence of rEPM. For B, C, and D, ≥20 colonies from each condition were examined. Bar, 100 μm.
Figure 4
Identification of ∼30-kD soluble EPM in vivo. (A) Frozen sections of prefixed (a) and unfixed (b) lactating mammary glands labeled with anti-EPM antibodies. The tissue in b was fixed on the slide immediately after sectioning, and the staining was carried out with mild washing so as not to remove soluble proteins in the lumina (asterisk). (B) Immunoblot analysis of the lactating mammary gland tissue (T) and milk (M) with anti–β-actin and anti-EPM antibodies. 5 (×5) or 1 μg protein samples of mammary gland extract (T) or milk (M) collected from lactating wild-type mice were probed with anti–β-actin (a) and EPM (b) antibodies. (C) 30-kD soluble EPM reacts with all anti-EPM antibodies except those directed against the COOH terminus. (a) Schematic diagram of affinity purified antibodies targeted to different domains of EPM. (b) Immunoblot analyses of milk and recombinant full-length 34-kD EPM (r-EPM) using the affinity purified antibodies. Anti-1, -2, -3, -1–230, and -c are specific to EPM amino acids 1–104, 105–188, 189–264, 1–230, and 231–264, respectively. Bar, 30 μm.
Figure 4
Identification of ∼30-kD soluble EPM in vivo. (A) Frozen sections of prefixed (a) and unfixed (b) lactating mammary glands labeled with anti-EPM antibodies. The tissue in b was fixed on the slide immediately after sectioning, and the staining was carried out with mild washing so as not to remove soluble proteins in the lumina (asterisk). (B) Immunoblot analysis of the lactating mammary gland tissue (T) and milk (M) with anti–β-actin and anti-EPM antibodies. 5 (×5) or 1 μg protein samples of mammary gland extract (T) or milk (M) collected from lactating wild-type mice were probed with anti–β-actin (a) and EPM (b) antibodies. (C) 30-kD soluble EPM reacts with all anti-EPM antibodies except those directed against the COOH terminus. (a) Schematic diagram of affinity purified antibodies targeted to different domains of EPM. (b) Immunoblot analyses of milk and recombinant full-length 34-kD EPM (r-EPM) using the affinity purified antibodies. Anti-1, -2, -3, -1–230, and -c are specific to EPM amino acids 1–104, 105–188, 189–264, 1–230, and 231–264, respectively. Bar, 30 μm.
Figure 5
Production of ∼30-kD soluble EPM in vitro. (A) The products of EPM cDNA tagged with T7 peptide at the NH2 terminus in transfected primary mammary cells (a), SCp2 cells (b), and SCp2′ cells (c) were analyzed by immunoblot with monoclonal anti-T7 antibody. Cells (C) and supernatants (S) were analyzed separately. For detection in the supernatant, an immunocomplex with anti-EPM antibodies was collected with protein A–Sepharose beads. Untagged rEPM was used as a control. (B) rEPM-tagged with 6× His at the NH2 terminus was incubated with membranes derived from lactating mammary glands (a) or from either SCp2 or SCp2′ cells (b). Products were collected with a Ni-agarose column and analyzed by immunoblotting with anti-EPM antibodies. For a, untagged rEPM was used as a control. In b, the MMP inhibitor GM6001 or the inactive structural homologue C1004 was added to a final concentration of 10 μM. Results are typical of three independent experiments.
Figure 5
Production of ∼30-kD soluble EPM in vitro. (A) The products of EPM cDNA tagged with T7 peptide at the NH2 terminus in transfected primary mammary cells (a), SCp2 cells (b), and SCp2′ cells (c) were analyzed by immunoblot with monoclonal anti-T7 antibody. Cells (C) and supernatants (S) were analyzed separately. For detection in the supernatant, an immunocomplex with anti-EPM antibodies was collected with protein A–Sepharose beads. Untagged rEPM was used as a control. (B) rEPM-tagged with 6× His at the NH2 terminus was incubated with membranes derived from lactating mammary glands (a) or from either SCp2 or SCp2′ cells (b). Products were collected with a Ni-agarose column and analyzed by immunoblotting with anti-EPM antibodies. For a, untagged rEPM was used as a control. In b, the MMP inhibitor GM6001 or the inactive structural homologue C1004 was added to a final concentration of 10 μM. Results are typical of three independent experiments.
Figure 6
WAP–EPM transgenic mice have enlarged ductal lumina and altered C/EBPβ expression. (A) Schematic diagram of the transgene construct. N, NotI; H, HindIII. (B) PCR analysis of the integration of the transgene into genomic DNA and RT-PCR analysis of EPM transgene expression. As a control for RT-PCR, endogenous stromelysin 1 (SL-1) was analyzed in the same samples. Lower bands (asterisk) are unreacted primers that remained in control samples after RT-PCR. (C) Phenotypic appearance of the mammary gland (circled) from midpregnant (7 d) normal (a) and transgenic (b) mice. Whole-mount stained mammary gland of normal (c) and transgenic (d) mice. (D) Analysis of C/EBPβ gene products in mammary tissue of normal and transgenic mice using commercial anti-C/EBPβ antibody. CRM, cross-reactive material. Bars: (B) 5 μm; (C) 300 μm.
Figure 6
WAP–EPM transgenic mice have enlarged ductal lumina and altered C/EBPβ expression. (A) Schematic diagram of the transgene construct. N, NotI; H, HindIII. (B) PCR analysis of the integration of the transgene into genomic DNA and RT-PCR analysis of EPM transgene expression. As a control for RT-PCR, endogenous stromelysin 1 (SL-1) was analyzed in the same samples. Lower bands (asterisk) are unreacted primers that remained in control samples after RT-PCR. (C) Phenotypic appearance of the mammary gland (circled) from midpregnant (7 d) normal (a) and transgenic (b) mice. Whole-mount stained mammary gland of normal (c) and transgenic (d) mice. (D) Analysis of C/EBPβ gene products in mammary tissue of normal and transgenic mice using commercial anti-C/EBPβ antibody. CRM, cross-reactive material. Bars: (B) 5 μm; (C) 300 μm.
Figure 6
WAP–EPM transgenic mice have enlarged ductal lumina and altered C/EBPβ expression. (A) Schematic diagram of the transgene construct. N, NotI; H, HindIII. (B) PCR analysis of the integration of the transgene into genomic DNA and RT-PCR analysis of EPM transgene expression. As a control for RT-PCR, endogenous stromelysin 1 (SL-1) was analyzed in the same samples. Lower bands (asterisk) are unreacted primers that remained in control samples after RT-PCR. (C) Phenotypic appearance of the mammary gland (circled) from midpregnant (7 d) normal (a) and transgenic (b) mice. Whole-mount stained mammary gland of normal (c) and transgenic (d) mice. (D) Analysis of C/EBPβ gene products in mammary tissue of normal and transgenic mice using commercial anti-C/EBPβ antibody. CRM, cross-reactive material. Bars: (B) 5 μm; (C) 300 μm.
Figure 6
WAP–EPM transgenic mice have enlarged ductal lumina and altered C/EBPβ expression. (A) Schematic diagram of the transgene construct. N, NotI; H, HindIII. (B) PCR analysis of the integration of the transgene into genomic DNA and RT-PCR analysis of EPM transgene expression. As a control for RT-PCR, endogenous stromelysin 1 (SL-1) was analyzed in the same samples. Lower bands (asterisk) are unreacted primers that remained in control samples after RT-PCR. (C) Phenotypic appearance of the mammary gland (circled) from midpregnant (7 d) normal (a) and transgenic (b) mice. Whole-mount stained mammary gland of normal (c) and transgenic (d) mice. (D) Analysis of C/EBPβ gene products in mammary tissue of normal and transgenic mice using commercial anti-C/EBPβ antibody. CRM, cross-reactive material. Bars: (B) 5 μm; (C) 300 μm.
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