Depletion of nuclear actin is a key mediator of quiescence in epithelial cells

Abstract

Functional differentiation is orchestrated by precise growth-regulatory controls conveyed by the tissue microenvironment. Cues from laminin 111 (LN1) lower transcription and suppress mammary epithelial cell growth in culture, but how LN1 induces quiescence is unknown. Recent literature points to involvement of nuclear β-actin in transcriptional regulation. Here, we show that quiescence induced by growth factor withdrawal, or LN1 addition, rapidly decreases nuclear β-actin. LN1, but not other extracellular matrix (ECM) molecules, decreases the levels of nuclear β-actin and destabilizes RNA polymerase (RNA Pol) II and III binding to transcription sites, leading to a dramatic drop in transcription and DNA synthesis. Constitutive overexpression of globular β-actin in the nucleus reverses the effect of LN1 on transcription and RNA Pol II association and prevents the cells from becoming quiescent in the presence of LN1. The physiological relevance of our findings was verified by identifying a clear spatial separation of LN1 and β-actin in developing mammary end buds. These data indicate a novel role for nuclear β-actin in growth arrest of epithelial cells and underscore the importance of the integrity of the basement membrane in homeostasis.

Fig. 1.

Growth and quiescence correlate with nuclear β-actin levels. (A) Mouse ScP2 cells were cultured under growth conditions for 48 hours and co-immunolabeled with antibodies against β-actin (green) and BrdU (red). The data show that, during S phase (represented by high BrdU incorporation), nuclei contain higher levels of actin than nuclei that are not undergoing DNA synthesis. Images are overlaid with white trace contours of corresponding DAPI images to delineate the limits of each nucleus. (B) 5-ethynyl-29-deoxyuridine (EdU) incorporation (i) and nuclear β-actin levels (ii) in ScP2 cells showing that, 48 hours after serum and growth factor removal, there is a significant and concomitant decrease in DNA synthesis and β-actin levels in Triton-extracted nuclei. For (i), the percentage of EdU-labeled nuclei represents the mean ± s.d. ratio of nuclei labeled with EdU to the total number of DAPI-stained nuclei in two independent experiments. Student's t-test (95% confidence interval). For (ii), nuclear β-actin levels were measured by western blot analysis on 5 μg of Triton-extracted nuclear protein and normalized to lamins A and C levels. The graph displays the mean ± s.d. change in nuclear β-actin levels from three experiments. Student's t-test (95% confidence interval). (C) BrdU incorporation in ScP2 cells showing a dramatic decline in the number of cells undergoing DNA synthesis after only 2 hours of laminin 111 (LN1) treatment in relation to the control (CNTRL). The percentage of BrdU-labeled nuclei represents the mean ± s.d. ratio of nuclei labeled with BrdU to the total number of DAPI-stained nuclei in two independent experiments. Student's t-test (95% confidence interval). (D) ScP2 cells were treated with laminin-rich ECM (LrECM) for up to 24 hours. Western blot analysis (top) with quantification (graph on bottom) shows that LrECM treatment dramatically decreases endogenous (endog.) nuclear β-actin by as early as 2 hours from the onset of treatment. Nuclear β-actin levels were measured by western blot analysis on 5 μg of Triton-extracted nuclear protein and normalized to the levels of lamins A and C. The graph displays the mean ± s.d. change in β-actin levels within Triton-extracted nuclear lysates from three experiments; Student's t-test (95% confidence interval). (E) EpH4 cells were transiently transfected with YFP–β-actin, and transgene expression levels were measured in response to treatment with LrECM over time (hours) as indicated. (F) EpH4 cells were transiently transfected with YFP, and transgene expression levels were measured in response to treatment with LrECM for 48 hours. Graphs in (E) and (F) display the mean ± s.d. change in fluorescence intensity levels within a 1 μm cytoplasmic (cyt) and nuclear (nuc) volume of treated relative to control (CNTRL) untreated cells from two or three experiments (n=100–154 cells per treatment). Statistical significance was calculated from the raw data by using a Student's t-test (95% confidence interval). Note that LrECM decreases nuclear and cytoplasmic YFP–β-actin, but not YFP alone, compared with control cells cultured in the absence of ECM for 24 hours and that nuclear β-actin levels start to decline significantly before cytoplasmic β-actin levels. NSD, no significant difference. Scale bars: 10 μm.

Fig. 2.

The levels of β-actin are high in the invading tip of terminal end buds and correlate inversely with LN1 localization. Sections of terminal end buds from pre-pubertal mouse mammary glands were labeled with (A) an antibody against β-actin or (B) fluorophore-tagged DNase, which binds to globular actin, and an antibody against the laminin 111 α 1 chain. Note that the spatial localization of DNase-bound actin is not as clear-cut as that shown with an antibody against β-actin in A. This is most likely because DNase can bind to all actin isoforms (β-, γ- and α-actin) (Garrels and Gibson, 1976) that are present in both the stromal and epithelial cells (indicated with white bracket and grey arrow). Nevertheless, there is still a clear separation between regions labeled with DNase and the LN1 antibody (please note the absence of yellow that would indicate colocalization of the two fluorophores). (C) EpH4 cells were transiently transfected with YFP–β-actin, and transgene expression levels were measured in response to treatment with Col IV, fibronectin (FN) or LN1 for 48 hours. Graphs display the mean ± s.d. change in fluorescence intensity levels within a 1 μm cytoplasmic (cyt) and nuclear (nuc) volume of treated relative to control cells from two or three experiments (n=22–154 cells per treatment). Statistical significance was calculated from the raw data by using a Student's t-test (95% confidence interval). Note that only LN1 significantly decreased nuclear and cytoplasmic YFP–β-actin compared with control cells. Scale bars: 50 μm (A,B); 10 μm (C).

Fig. 3.

Constitutive expression of a NLS–β-actin or NLS–β-actin R62D transgene opposes LN1-mediated growth arrest. (A) EpH4 cells were stably transfected with FLAG-tagged NLS–wild-type-β-actin. Clones expressing high- and low-transgene levels were expanded and immunolabeled with antibody against FLAG. The images represent single nuclei from each clonal population and are overlaid with white trace contours of corresponding DAPI images to delineate the limits of each nucleus. Scale bar: 10 μm. (B) Measurement of BrdU incorporation after 2 hours of LN1 treatment showed that the percentage of cells incorporating BrdU was twice as great in clones expressing high versus low levels of NLS-wild-type (left) or R62D mutant globular (right) β-actin. The percentage incorporation represents the mean ± s.d. ratio of nuclei labeled with BrdU to the total number of DAPI-stained nuclei in two independent experiments. Student's t-test (95% confidence interval).

Fig. 4.

There is a statistically significant colocalization of nuclear β-actin with transcription foci. (A) Immunofluorescence localization of FU incorporation sites in a tissue section of a terminal end bud from pre-pubertal mouse mammary gland. Note that the localization of F-RNA is similar to globular β-actin, but both are inversely correlated with the LN1 localization shown in Fig. 2B. (B) (i) Nuclei from ScP2 cells cultured under growth conditions were analyzed by immunofluorescence for endogenous (endog.) β-actin colocalization with F-RNA (left). (ii) Graph displays each nucleus as a circle and the percentage of β-actin nonrandomly colocalized with F-RNA per nucleus (yellow in image; n=39). All colocalization values were calculated as described in the Materials and Methods section and are statistically significant (P<0.05). (C,D) Quantification of F-RNA incorporation in ScP2 cells in response to LrECM treatment over time showing that LrECM treatment causes a dramatic decline in transcriptional activity over 24 hours compared with that of control cells. Graphs represent: (C) the mean ± s.d. pixel intensity and (D) nuclear volume of F-RNA transcription foci over LrECM treatment time (n=64–114 nuclei per time-point). Values are representative of two independent experiments. Abbreviation: a.u., arbitrary units. Student's t-test (95% confidence interval). (E) F-RNA incorporation in cells treated for 48 hours with LrECM, collagen IV (Col IV) or laminin 111 (LN1) showing that it is the LN1 component of LrECM that represses transcriptional activity. Images are overlaid with white trace contours of corresponding DAPI images to delineate the limits of each nucleus. Scale bars: 50 μm (A); 2 μm (B); 10 μm (E).

Fig. 5.

Constitutive expression of a NLS–β-actin R62D transgene opposes LN1-mediated transcriptional repression. (A) (i) Nuclei from ScP2 cells cultured under growth conditions were analyzed for colocalization of NLS–β-actin with RNA Pol II (left; scale bar: 2 μm). (ii) The graph displays each nucleus as a circle and the percentage of NLS–β-actin nonrandomly colocalized with RNA Pol II per nucleus (yellow in image; n=26). All colocalization values were calculated as described in the Materials and Methods section and are statistically significant (P<0.05). (B) Cells were transiently transfected with NLS-vector or NLS–β-actin R62D and then treated with LN1 for 24 hours. After treatment, individual nuclei were subjected to an in vitro nuclear run-on assay, immunostained for FU and then analyzed for the percentage of nuclear volume occupied by F-RNA. The data indicate that the nuclei of cells expressing NLS–β-actin contain a larger volume of F-RNA than the nuclei of cells expressing the NLS vector control. A Mann–Whitney test was used to calculate all P values.

Fig. 6.

LN1 treatment destabilizes RNA Pol II and III interactions with nuclear substructures. (A) RNA Pol III and (B) RNA Pol II levels in Triton-extracted nuclei of cells treated with LrECM for the durations indicated were detected by western blot analysis. Graphs display the mean ± s.d. from two or three experiments for RNA polymerase levels in 20 μg of Triton-resistant nuclear protein preparations after normalization to the levels of lamins A and C; Student's t-test (95% confidence interval). Note that LN1 treatment destabilizes RNA Pol III and II association with the nuclear substructure within 2 and 4 hours of LrECM treatment, respectively; Student's t-test (95% confidence interval). (C) Cells were transiently transfected with NLS vector or NLS–β-actin R62D and then treated with LN1 for 24 hours. After treatment, individual nuclei were analyzed for the percentage of nuclear volume occupied by Triton-resistant RNA Pol II (II_O and II_A). The data indicate that the nuclei of cells expressing NLS–β-actin contain a larger volume of Triton-resistant RNA Pol II than the nuclei of cells expressing the NLS vector control. A Mann–Whitney test was used to calculate all P values.

Fig. 7.

Schematic model of the sequence of LN1-initiated events that give rise to quiescence. (A) Exposure of mammary epithelial cells to LN1 induces a dramatic decline in the levels of nuclear β-actin, nuclear matrix (NM)-associated RNA Pol III and DNA synthesis by 2 hours. By 4 hours, the levels of both NM-bound RNA Pol II and cytoplasmic β-actin also begin to decline. It is important to note that these pools are not completely depleted. The populations of nuclear β-actin, Pol II and Pol III remaining after growth arrest are probably required for LN1-induced tissue-specific gene expression. CNTRL, control. (B) Working model showing that LN1 engagement with cell surface receptors such as dystroglycan (DG) or integrin (Int) influences phosphoinositide 3-kinase (PI3K) activity in a manner that decreases nuclear β-actin levels, DNA and RNA synthesis and RNA Pol III binding to the nuclear matrix. These events would work in concert to decrease the level of nuclear-matrix-associated RNA Pol II, resulting in an additional decrease in RNA synthesis that is either contributory to, coincidental with or a consequence of a reduction in cytosolic β-actin levels. The nature of the detailed interactions between these events remains to be elucidated. FAK, focal adhesion kinase.