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First published online July 31, 2003
doi: 10.1242/10.1242/jcs.00656

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ECM-induced gap junctional communication enhances mammary epithelial cell differentiation

¹ Department of Human Morphology, Faculty of Medicine
² Department of Biology, Faculty of Arts and Sciences, American University of Beirut, PO Box 11-0236, Beirut, Lebanon

View larger version (91K): [in a new window]   Fig. 1. ECM affects morphology, ß-casein and connexin expression by CID-9 cells on day 6 of culture. (A) Morphology of CID-9 cells cultured in differentiation medium on (a) plastic, (b) EHS-drip and (c) EHS-matrix. (B) Northern blot analysis of ß-casein, Cx43 and Cx26 by cells on plastic (Pl), EHS-drip (Ed) and EHS-matrix (Ec). ß-Casein expression was only evident in the presence (+) of prolactin (Prl) and not in its absence (-), whereas Cx43 was significantly (P<0.001) downregulated in cells in differentiation medium on Ec compared with cells on Pl or Ed. Cx26 was not downregulated in cells in differentiation medium on Ec compared with those on Pl. 28S rRNA demonstrates equal loading.

View larger version (42K): [in a new window]   Fig. 2. ECM modulates connexin protein expression and localization of CID-9 cells cultured in differentiation medium on day 6 of culture. (A) Western blot analysis of Cx26, Cx32 and Cx43. Cells were plated on plastic (Pl), EHS-drip (Ed) or EHS-matrix (Ec). P2, P1 and NP denotes the different phosphorylation states of Cx43. Cx43 P2 form was signifcanlty (P<0.001) upregulated in cells on Ec compared with those on Pl or Ed. (B) Immunolocalization of connexins in CID-9 cells, plated on plastic (a,c,e) and on EHS-drip (b,d,f). Cells were immunostained for Cx26 (a,b), Cx32 (c,d) and Cx43 (e,f). Note predominant membranous localization of connexins in cells cultured on EHS-drip (b,d,f). Inserts in (a), (c) and (e) show occasional membranous localization noted in cells cultured on plastic.

View larger version (97K): [in a new window]   Fig. 3. ECM enhances GJIC. Microinjection (A,C) and scrape loading (B,D) of LY in CID-9 cells on day 6 of culture. Cells were plated in differentiation medium on plastic (A,B) or on EHS-drip (C,D). The bar corresponds to about 2 cells in (A) and about 10 cells in (C). Integrated fluorescence intensity, quantified from three different LY scrape-load experiments as described in Materials and Methods, showed a threefold decrease between cells on EHS-drip (Drip) and on plastic (Pl).

View larger version (41K): [in a new window]   Fig. 4. 18 GA inhibits GJIC and downregulates ß-casein expression by CID-9 cells on day 5 of culture in differentiation medium. (A) Cells were plated on EHS-drip in the absence (Ctrl; control) or presence of 10 µM 18 GA (18G). LY dye transfer in scrape-loaded cells was markedly affected by 18 GA. Integrated fluorescence intensity, quantified from three different experiments as described in Materials and Methods, showed a threefold decrease. Western blot analysis showed that 18 GA downregulated Cx43 and ß-casein levels without drastically affecting cell viability as determined by Trypan Blue staining. (B) Morphology of CID-9 cells plated in differentiation medium on EHS-matrix on day 6 of culture (a), compared with cells treated with 10 µM 18 GA (b). Note the partial restoration of normal clustering morphology 4 days (i.e. day 10 of culture) after 18 GA removal (c). Western blot analysis, normalized to equal cell count, showed that ß-casein was partially recovered (approximately 50% recovery) upon 18 GA withdrawal.

View larger version (38K): [in a new window]   Fig. 5. cAMP enhances GJIC, ß-casein and connexin expression by CID-9 cells on day 6 of culture in differentiation medium. Cells were plated on plastic in the absence (Ctrl; control) or presence of 50 µM 8-Br-cAMP (cAMP). (A) LY dye transfer in scrape-loaded cells was enhanced by cAMP. Integrated fluorescence intensity, quantified from three different experiments as described in Materials and Methods, showed a 3.2-fold increase (P<0.01). Phase contrast light micrograph showing altered clustering behaviour in cells treated with cAMP. (B) Northern blot analysis of ß-casein for cells plated on (Ctrl) plastic, and (cAMP) plastic plus 50 µM 8-Br-cAMP. 28S rRNA demonstrates equal loading. (C) Western blot for ß-casein, Cx43, Cx26 and ß-actin for CID-9 cells plated on (Ctrl) plastic, and (cAMP) plastic plus 50 µM 8-Br-cAMP. ß-actin western blot demonstrates equal loading. Western blot analysis showed a significant increase in ß-casein (P<0.01), Cx43 (P<0.05) and Cx26 (P<0.001) levels.

View larger version (32K): [in a new window]   Fig. 6. cAMP induces ß-casein expression in a GJIC-dependent and ß1-integrin-independent pathway. (A) Western blot analysis for ß-casein and Cx43, for CID-9 cells plated on (1) plastic, (2) plastic plus 50 µM 8-Br-cAMP, (3) plastic plus 50 µM 8-Br-cAMP and 10 µM 18 GA. ß-actin western blot demonstrates equal loading in (1), (2) and (3). Graph insets show that 8-Br-cAMP-treated cells on plastic had significantly higher ß-casein (P<0.05) and Cx43 (P<0.01) levels than untreated cells on plastic, whereas 18 GA significantly downregulated ß-casein (P<0.01) and Cx43 (P<0.01) levels in cells on plastic treated with 8-Br-cAMP. (B) Western blot analysis for ß-casein expression by CID-9 cells plated on (1) plastic plus 50 µM 8-Br-cAMP, (2) EHS-drip, (3) plastic plus 50 µM 8-Br-cAMP and 100 µg/ml anti-integrin, and (4) EHS-drip plus 100 µg/ml anti-integrin. ß-actin western blot was used to quantify ß-casein levels in (1), (2), (3) and (4) after normalization to equal ß-actin loading. A significant decrease in ß-casein levels (P<0.01) is noted when cells on EHS-drip are treated with anti-integrin antibody (4) compared with untreated cells (2). This is not the case when cells on plastic and supplemented with 8-Br-cAMP (1) are treated with the same antibody (3).

View larger version (85K): [in a new window]   Fig. 7. cAMP induces ß-casein expression in an adhesion-independent manner. (A) Morphology of CID-9 cells on day 6 of culture, plated on PolyHEMA (Ctrl; control) and supplemented with 50 µM 8-Br-cAMP (cAMP). (B) Western blot for ß-casein, Cx43, Cx32 and Cx26 and for ß-actin for cells plated on PolyHEMA (Ctrl; control) and supplemented with 50 µM 8-Br-cAMP (cAMP). Significant increase in ß-casein expression (P<0.001) and Cx43 (P<0.001) was noted when cells on polyHEMA were treated with 8-Br-cAMP compared with untreated cells.