Differentiation-Dependent Alternative Splicing and Expression of the Extracellular Matrix Protein 1 Gene in Human Keratinocytes  Patrick Smits, Yves Poumay, Marcel Karperien, Przemko Tylzanowski, Jan Wauters, Danny Huylebroeck, Maria Ponec, Jozef Merregaert  Journal of Investigative Dermatology  Volume 114, Issue 4, Pages 718-724 (April 2000) DOI: 10.1046/j.1523-1747.2000.00916.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Ecm1b mRNA is expressed in differentiated keratinocytes. (A) Northern blot analysis of Ecm1 expression in differentiating human keratinocyte cultures. Normal human keratinocytes were grown in autonomous conditions and induced to differentiate by manipulating the cell density. Poly(A+)RNA was extracted (lane 1) when cells were covering 70%-80% of the culture disk, or (lane 2) approximately 4 d later when keratinocytes had reached confluency, and (lane 3) 7 d after confluency, and was subjected to northern blot analysis. Blots were hybridized with the full length human Ecm1a cDNA probe and keratin K10, K14, and involucrin probes. RNA loading was checked by hybridization with the housekeeping gene cyclophilin. (B) Northern blot analysis of keratinocyte cultures treated with TPA. Subconfluent normal human keratinocytes were treated with 0 ng TPA per ml (lane 1), 1 ng TPA per ml (lane 2), and 10 ng TPA per ml (lane 3) for 18 h. Poly(A+)RNA was extracted and subjected to northern blot analysis. Journal of Investigative Dermatology 2000 114, 718-724DOI: (10.1046/j.1523-1747.2000.00916.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Ecm1a and Ecm1b mRNAs have a complementary expression pattern in adult human epidermis. (A) Bright field picture of in situ hybridization on normal human adult epidermis with an antisense 35S-labeled cRNA probe reacting specifically with Ecm1a mRNA. This probe does not cross-react with Ecm1b mRNAs. (B) Bright field picture of in situ hybridization on normal human adult epidermis with the sense 35S-labeled control cRNA probe of the probe used in (A). (C) Bright field picture of in situ hybridization on normal human adult epidermis with an antisense 35S-labeled cRNA probe reacting with both Ecm1a and Ecm1b mRNAs transcripts. (D) Bright field picture of in situ hybridization on normal human adult epidermis with the corresponding sense 35S-labeled control cRNA probe of the probe used in (C). Magnification: 100× for all figures. Journal of Investigative Dermatology 2000 114, 718-724DOI: (10.1046/j.1523-1747.2000.00916.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Ecm1a and Ecm1b proteins have a complementary expression pattern in adult human epidermis. (A) Peroxidase staining of normal human adult epidermis using the sp23 antibody specifically reacting with Ecm1a. (B) Peroxidase staining of normal human adult epidermis using the OAP 12516 antibody recognizing both Ecm1a and Ecm1b. B, basal layer; E, epidermis; D, dermis. Weaker staining in the dermis is indicated by arrows. Journal of Investigative Dermatology 2000 114, 718-724DOI: (10.1046/j.1523-1747.2000.00916.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Ecm1 is localized centromeric at a distance of 2 Mbp from the EDC at chromosome 1q21. FISH was performed using three different probes: (i) cosmid clone ICRFc112L1566 containing the human macrophage colony stimulating factor gene (red), which is mapped at 1p13–21; (ii) YAC clone y767A1 containing the centromeric part of the EDC (green); and (iii) cosmid clone D0860Q containing the human Ecm1 gene (yellow). (A) A methaphase spread of chromosomes 1; (B) an interphase spread. Journal of Investigative Dermatology 2000 114, 718-724DOI: (10.1046/j.1523-1747.2000.00916.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Ecm1b mRNA transcription can be regulated by coculture with stromal cells. (A) Normal human keratinocytes were grown in the absence (lane 1) or the presence of lethally irradiated human dermal fibroblasts (lane 2), NIH 3T3 (lane 3), or MC 3T3 cells (lane 4). Total RNA was extracted and analyzed by northern blotting using a full length human Ecm1a cDNA probe. Blots were hybridized with a 28S rRNA probe to control for RNA loading. (B) Normal human keratinocytes were seeded on an inert filter and cultured for 8 d. Human dermal fibroblasts, NIH 3T3, and MC 3T3 cells were seeded in parallel dishes. Filters were subsequently transferred into six well plates on the bottom of which no cells or fibroblastic cells were present. Cocultures were incubated for the next 2 d after which total RNA of epithelial cells and fibroblasts was harvested and subjected to northern blot analysis. Hybridizations were performed for human material (NHK, dermal fibroblasts, SCC-4) with human full length Ecm1a cDNA probes, and for mouse material (NIH 3T3, MC 3T3) with the mouse full length Ecm1a cDNA as probe. Hybridization with 28S probe was performed to control for RNA loading. In lanes 1, 3, 5, and 7, NHK were cultured in inserts with no cells (lane 1), human dermal fibroblasts (lane 3), NIH 3T3 (lane 5), or MC 3T3 cells (lane 7) at the bottom. In lanes 2, 4, and 6 human dermal fibroblasts, NIH 3T3, and MC 3T3 cells, respectively, were cultured without inserts. (C) An experiment similar to (B) except that SCC-4 cells instead of NHK were used. In lanes 1, 3, 5, and 7 SCC-4 cells were cultured in inserts with no cells (lane 1), human dermal fibroblasts (lane 3), NIH 3T3 (lane 5), or MC 3T3 cells (lane 7) at the bottom. In lanes 2, 4, and 6, human dermal fibroblasts, NIH 3T3, and MC 3T3 cells, respectively, were cultured without inserts. Journal of Investigative Dermatology 2000 114, 718-724DOI: (10.1046/j.1523-1747.2000.00916.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions