Identification of a ZEB2-MITF-ZEB1 transcriptional network that controls melanogenesis and melanoma progression.

Deregulation of signaling pathways that control differentiation, expansion and migration of neural crest-derived melanoblasts during normal development contributes also to melanoma progression and metastasis. Although several epithelial-to-mesenchymal (EMT) transcription factors, such as zinc finger E-box binding protein 1 (ZEB1) and ZEB2, have been implicated in neural crest cell biology, little is known about their role in melanocyte homeostasis and melanoma. Here we show that mice lacking Zeb2 in the melanocyte lineage exhibit a melanoblast migration defect and, unexpectedly, a severe melanocyte differentiation defect. Loss of Zeb2 in the melanocyte lineage results in a downregulation of the Microphthalmia-associated transcription factor (Mitf) and melanocyte differentiation markers concomitant with an upregulation of Zeb1. We identify a transcriptional signaling network in which the EMT transcription factor ZEB2 regulates MITF levels to control melanocyte differentiation. Moreover, our data are also relevant for human melanomagenesis as loss of ZEB2 expression is associated with reduced patient survival.

Smad-interacting protein 1 affects acute and tonic, but not chronic pain.

BACKGROUND: Smad-interacting protein 1 (also named Zeb2 and Zfhx1b) is a transcription factor that plays an important role in neuronal development and, when mutated, causes Mowat-Wilson syndrome (MWS). A corresponding mouse model carrying a heterozygous Zeb2 deletion was comprehensively analysed in the German Mouse Clinic. The most prominent phenotype was the reduced pain sensitivity. In this study, we investigated the role of Zeb2 in inflammatory and neuropathic pain. METHODS: For this, we tested mutant Zeb2 animals in different models of inflammatory pain like abdominal constriction, formalin and carrageenan test. Furthermore, we studied the pain reactivity of the mice after peripheral nerve ligation. To examine the nociceptive transmission of primary sensory dorsal root ganglia (DRG) neurons, we determined the neuronal activity in the spinal dorsal horn after the formalin test using staining of c-Fos. Next, we characterized the neuronal cell population in the DRGs and in the sciatic nerve to study the effect of the Zeb2 mutation on peripheral nerve morphology. RESULTS: The present data show that Zeb2 is involved in the development of primary sensory DRG neurons, especially of C- and Aδ fibres. These alterations contribute to a hypoalgesic phenotype in inflammatory but not in neuropathic pain in these Zeb2(+/-) mice. CONCLUSION: Our data suggest that the under-reaction to pain observed in MWS patients results from a reduced responsivity to nociceptive stimulation rather than an inability to communicate discomfort.

Functional redundancy of TGF-beta family type I receptors and receptor-Smads in mediating anti-Mullerian hormone-induced Mullerian duct regression in the mouse.

Amniotes, regardless of genetic sex, develop two sets of genital ducts: the Wolffian and Müllerian ducts. For normal sexual development to occur, one duct must differentiate into its corresponding organs, and the other must regress. In mammals, the Wolffian duct differentiates into the male reproductive tract, mainly the vasa deferentia, epididymides, and seminal vesicles, whereas the Müllerian duct develops into the four components of the female reproductive tract, the oviducts, uterus, cervix, and upper third of the vagina. In males, the fetal Leydig cells produce testosterone, which stimulates the differentiation of the Wolffian duct, whereas the Sertoli cells of the fetal testes express anti-Müllerian hormone, which activates the regression of the Müllerian duct. Anti-Müllerian hormone is a member of the transforming growth factor-beta (TGF-beta) family of secreted signaling molecules and has been shown to signal through the BMP pathway. It binds to its type II receptor, anti-Müllerian hormone receptor 2 (AMHR2), in the Müllerian duct mesenchyme and through an unknown mechanism(s); the mesenchyme induces the regression of the Müllerian duct mesoepithelium. Using tissue-specific gene inactivation with an Amhr2-Cre allele, we have determined that two TGF-beta type I receptors (Acvr1 and Bmpr1a) and all three BMP receptor-Smads (Smad1, Smad5, and Smad8) function redundantly in transducing the anti-Müllerian hormone signal required for Müllerian duct regression. Loss of these genes in the Müllerian duct mesenchyme results in male infertility due to retention of Müllerian duct derivatives in an otherwise virilized male.

Functional redundancy of extracellular matrix protein 1 in epidermal differentiation.

BACKGROUND: Extracellular matrix protein 1 (ECM1) is a secreted protein expressed in skin. Its dermatological relevance has been highlighted by the discovery of loss-of-function mutations in ECM1 in patients with lipoid proteinosis (LiP). OBJECTIVES: To determine the role of ECM1 in epidermal differentiation by examining gene and protein expression of epidermal differentiation markers in individuals with LiP and histological assessment of transgenic mouse skin that overexpresses Ecm1a in basal or suprabasal epidermis. METHODS: Subconfluent, confluent and postconfluent LiP and control keratinocyte cultures were analysed by Northern and Western blotting for differences in expression of differentiation markers. Expression of these markers was analysed in skin of patients with LiP by immunohistochemistry. To study effects of Ecm1 overexpression on epidermal differentiation, transgenic mice were generated under control of either a keratin 14 or an involucrin promoter. RESULTS: No differential expression of the different markers analysed was observed in LiP keratinocytes compared with controls. No histological differences were found in Ecm1-overexpressing mouse skin compared with wild-type. CONCLUSIONS: Absence of ECM1 does not lead to differences in epidermal differentiation. Moreover, overexpression of Ecm1a in vivo does not exert dramatic effects on epidermal structure. Collectively, these findings suggest no role of ECM1 in epidermal differentiation.

NF-kappaB is involved in the regulation of CD154 (CD40 ligand) expression in primary human T cells.

Cognate interactions between CD154 (CD40 ligand, CD40L) on activated T cells and its receptor CD40 on various antigen-presenting cells are involved in thymus-dependent humoral immune responses and multiple other cell-mediated immune responses. We have studied the regulation of CD154 expression in human T cells after activation with anti-CD3 and anti-CD28 antibodies or after pharmacological activation of protein kinase C with phorbol 12-myristate 13-acetate, and the calcium ionophore ionomycin. Under these conditions, transcription of the CD154 gene was rapidly induced without requiring de novo protein synthesis. Pharmacological inhibitors of NF-kappaB activation down-regulated CD154 mRNA and protein levels. Cyclosporin A, an inhibitor of NF-AT activation, acted similarly, and the effects of both inhibitors were additive. A potential NF-kappaB binding site is present within the CD154 promoter at positions -1190 to - 1181. In electrophoretic mobility shift assays, this sequence was specifically bound by NF-kappaB present in nuclear extracts from activated T cells. Furthermore, in transient co-transfection of Jurkat T cells, p65 activated the transcription of a reporter construct containing a multimer of this NF-kappaB binding site. These observations demonstrate a role of NF-kappaB transcription factors in the regulation of CD40L expression in activated primary human T cells.

Analysis of Schizosaccharomyces pombe mediator reveals a set of essential subunits conserved between yeast and metazoan cells.

With the identification of eight new polypeptides, we here complete the subunit characterization of the Schizosaccharomyces pombe RNA polymerase II holoenzyme. The complex contains homologs to all 10 essential gene products present in the Saccharomyces cerevisiae Mediator, but lacks clear homologs to any of the 10 S. cerevisiae components encoded by nonessential genes. S. pombe Mediator instead contains three unique components (Pmc2, -3, and -6), which lack homologs in other cell types. Presently, pmc2(+) and pmc3(+) have been shown to be nonessential genes. The data suggest that S. pombe and S. cerevisiae share an essential protein module, which associates with nonessential speciesspecific subunits. In support of this view, sequence analysis of the conserved yeast Mediator components Med4 and Med8 reveals sequence homology to the metazoan Mediator components Trap36 and Arc32. Therefore, 8 of 10 essential genes conserved between S. pombe and S. cerevisiae also have a metazoan homolog, indicating that an evolutionary conserved Mediator core is present in all eukaryotic cells. Our data suggest a closer functional relationship between yeast and metazoan Mediator than previously anticipated.

Smad-interacting protein 1 is a repressor of liver/bone/kidney alkaline phosphatase transcription in bone morphogenetic protein-induced osteogenic differentiation of C2C12 cells.

Up-regulation of liver/bone/kidney alkaline phosphatase (LBK-ALP) has been associated with the onset of osteogenesis in vitro. Its transcription can be up-regulated by bone morphogenetic proteins (BMPs), constitutively active forms of their cognate receptors, or appropriate Smads. The promoter of LBK-ALP has been characterized partially, but not much is known about its transcriptional modulation by BMPs. A few Smad-interacting transcriptional factors have been isolated to date. One of them, Smad-interacting protein 1 (SIP1), belongs to the family of two-handed zinc finger proteins binding to E2-box sequences present, among others, in the promoter of mouse LBK-ALP. In the present study we investigated whether SIP1 could be a candidate regulator of LBK-ALP transcription in C2C12 cells. We demonstrate that SIP1 can repress LBK-ALP promoter activity induced by constitutively active Alk2-Smad1/Smad5 and that this repression depends on the binding of SIP1 to the CACCT/CACCTG cluster present in this promoter. Interestingly, SIP1 and alkaline phosphatase expression domains in developing mouse limb are mutually exclusive, suggesting the possibility that SIP1 could also be involved in the transcriptional regulation of LBK-ALP in vivo. Taken together, these results offer an intriguing possibility that ALP up-regulation at the onset of BMP-induced osteogenesis could involve Smad/SIP1 interactions, resulting in the derepression of that gene.

Transforming growth factor beta signalling in vitro and in vivo: activin ligand-receptor interaction, Smad5 in vasculogenesis, and repression of target genes by the deltaEF1/ZEB-related SIP1 in the vertebrate embryo.

The identification and characterization of components of the transforming growth factor beta (TGFbeta) signalling pathway are proceeding at a very fast pace. To illustrate a number of our activities in this field, we first summarize our work aiming at the selection from a large collection of single residue substitution mutants of two activin A polypeptides in which D27 and K102, respectively, have been modified. This work has highlighted the importance of K102 and its positive charge for binding to activin type II receptors. Activin K102E, which did not bind to high-affinity receptor complexes, may be a valuable beta chain, when incorporated in recombinant inhibin to unambiguously detect novel inhibin binding sites at the cell surface. We then illustrate how Smad5 knockout mice and an overexpression approach with a truncated TGFbeta type II receptor in the mouse embryo can contribute to the identification of a novel TGFbeta-->TbetaRII/ALK1-->Smad5 pathway in endothelial cells in the embryo proper and the yolk sac vasculature. We conclude with a summary of our results with a Smad-interacting transcriptional repressor but focus on its biological significance in the vertebrate embryo.

The two-handed E box binding zinc finger protein SIP1 downregulates E-cadherin and induces invasion.

Transcriptional downregulation of E-cadherin appears to be an important event in the progression of various epithelial tumors. SIP1 (ZEB-2) is a Smad-interacting, multi-zinc finger protein that shows specific DNA binding activity. Here, we report that expression of wild-type but not of mutated SIP1 downregulates mammalian E-cadherin transcription via binding to both conserved E2 boxes of the minimal E-cadherin promoter. SIP1 and Snail bind to partly overlapping promoter sequences and showed similar silencing effects. SIP1 can be induced by TGF-beta treatment and shows high expression in several E-cadherin-negative human carcinoma cell lines. Conditional expression of SIP1 in E-cadherin-positive MDCK cells abrogates E-cadherin-mediated intercellular adhesion and simultaneously induces invasion. SIP1 therefore appears to be a promoter of invasion in malignant epithelial tumors.

Extracellular matrix protein 1 (ECM1) has angiogenic properties and is expressed by breast tumor cells.

Tumor growth and metastasis are critically dependent on the formation of new blood vessels. The present study found that extracellular matrix protein 1 (ECM1), a newly described secretory glycoprotein, promotes angiogenesis. This was initially suggested by in situ hybridization studies of mouse embryos indicating that the ECM1 message was associated with blood vessels and its expression pattern was similar to that of flk-1, a recognized marker for endothelium. More direct evidence for the role of ECM1 in angiogenesis was provided by the fact that highly purified recombinant ECM1 stimulated the proliferation of cultured endothelial cells and promoted blood vessel formation in the chorioallantoic membrane of chicken embryos. Immunohistochemical staining with specific antibodies indicated that ECM1 was expressed by the human breast cancer cell lines MDA-435 and LCC15, both of which are highly tumorigenic. In addition, staining of tissue sections from patients with breast cancer revealed that ECM1 was present in a significant proportion of primary and secondary tumors. Collectively, the results of this study suggest that ECM1 possesses angiogenic properties that may promote tumor progression.

Mice with a homozygous gene trap vector insertion in mgcRacGAP die during pre-implantation development.

In a phenotypic screen in mice using a gene trap approach in embryonic stem cells, we have identified a recessive loss-of-function mutation in the mgcRacGAP gene. Maternal protein is present in the oocyte, and mgcRacGAP gene transcription starts at the four-cell stage and persists throughout mouse pre-implantation development. Total mgcRacGAP deficiency results in pre-implantation lethality. Such E3.5 embryos display a dramatic reduction in cell number, but undergo compaction and form a blastocoel. At E3.0-3.5, binucleated blastomeres in which the nuclei are partially interconnected are frequently observed, suggesting that mgcRacGAP is required for normal mitosis and cytokinesis in the pre-implantation embryo. All homozygous mutant blastocysts fail to grow out on fibronectin-coated substrates, but a fraction of them can still induce decidual swelling in vivo. The mgcRacGAP mRNA expression pattern in post-implantation embryos and adult mouse brain suggests a role in neuronal cells. Our results indicate that mgcRacGAP is essential for the earliest stages of mouse embryogenesis, and add evidence that CYK-4-like proteins also play a role in microtubule-dependent steps in the cytokinesis of vertebrate cells. In addition, the severe phenotype of null embryos indicates that mgcRacGAP is functionally non-redundant and cannot be substituted by other GAPs during early cleavage of the mammalian embryo.

SIP1 (Smad interacting protein 1) and deltaEF1 (delta-crystallin enhancer binding factor) are structurally similar transcriptional repressors.

BACKGROUND: Smad proteins are intracellular mediators of transforming growth factor-beta (TGFbeta) signalling that regulate gene expression by interacting with different classes of transcription factors including DNA-binding multi-zinc finger proteins. One of these, Smad interacting protein 1 (SIP1), is a novel two-handed zinc-finger protein that displays strong similarity with the transcriptional repressor delta-crystallin enhancer binding factor (deltaEF1). Here, we summarize what is known about the mechanism of action of both proteins and their role in vertebrate embryogenesis. Our data are discussed together with the present knowledge on other zinc-finger containing Smad interacting proteins. METHODS: The activities and function of SIP1 have been analysed through documentation of expression patterns, the effect of over-expression of SIP1 on target-gene expression, and promoter studies using Xenopus embryos. Moreover, S1P1/Smad complexes and their association with target promoter DNA were analyzed in biochemical studies. RESULTS: SIP1 is a transcriptional repressor displaying overlapping DNA binding specificities with deltaEF1. An in vivo target of SIP1 in Xenopus is a gene required for the formation of mesoderm, Brachyury (XBra). Our data indicate that SIP1 is required to confine XBra gene expression to the mesoderm. Furthermore, the expression pattern in Xenopus invites us to speculate that SIP1 plays a role in specification/differentiation of neuroectoderm. Unlike deltaEF1, SIP1 can bind directly to activated receptor regulated Smads (R-Smads) and recruit them to the DNA. This indicates that Smads may modulate the activity of SIP1 as a transcriptional repressor. CONCLUSIONS: Our data point to a role of SIP1 in developmental processes regulated by members of the TGFbeta family such as induction of mesoderm (mediated through activin-like signalling) and inhibition of neuroectoderm formation (mediated by bone morphogenetic proteins [BMPs]). Whereas SIP1 could act in TGFbeta signal transduction by virtue of interaction with activated R-Smads, genetic studies in the mouse indicate that deltaEF1 may act in certain TGFbeta pathways-i.e., BMPs and growth and differentiation factors (GDFs)-as well. The molecular mechanisms by which these transcriptional repressors act, as well as the function of the SIP1/Smad interaction, remain to be elucidated. CLINICAL RELEVANCE: Mutations in components of the TGFbeta signalling pathways have been associated with disease and congenital malformations. We anticipate that identification of Smad interacting transcription factors including SIP1 and their targets will help us to understand the molecular basis of certain pathologies.

Expression of the follistatin/EGF-containing transmembrane protein M7365 (tomoregulin-1) during mouse development.

A novel transmembrane protein (designated X7365) containing two follistatin modules and an epidermal growth factor (EGF) domain has been described in the hypothalamic-pituitary axis of Xenopus laevis. We have now cloned the highly conserved mouse orthologue (M7365), and its mRNA was detected in many mesodermal and (neuro)ectodermal tissues in 8.5-day-old mouse embryos. During further development, M7365 mRNA expression became restricted to certain regions in the brain and to ganglia. In the adult mouse, the brain is the major site of M7365 expression.

The bone morphogenetic protein 2 signaling mediator Smad1 participates predominantly in osteogenic and not in chondrogenic differentiation in mesenchymal progenitors C3H10T1/2.

The role of the bone morphogenetic protein (BMP)-signaling mediator Smad1 in osteogenic or chondrogenic differentiation was investigated in murine parental mesenchymal progenitors C3H10T1/2 and its derivatives constitutively expressing BMP-2 (C3H10T1/2-BMP-2) and, therefore, undergo BMP-mediated osteogenic/ chondrogenic development. The functions of the three Smad1 domains, that is, the N-terminal (MH1) domain, the C-terminal (MH2) domain, and the midregional proline-rich linker domain, were documented and compared with full-length Smadl. We showed that expression of the MH2 domain in parental C3H10T1/2 cells was sufficient to initiate osteogenic differentiation. Interestingly, MH1 was sufficient to initiate transcription of osteogenic marker genes like the osteocalcin or parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) receptor. However, MH1 interfered with the histologically distinct formation of osteoblast-like cells. A dominant-negative effect on MH2-mediated osteogenic development in C3H10T1/2 cells was observed by the dose-dependent trans-expression of the midregional linker domain. Importantly, in contrast to osteogenic differentiation, Smad1 and its domains do not mimic or interfere with BMP-2-dependent chondrogenic development as monitored by the inability of MH2 to give rise to histologically distinct chondrocytes in parental C3H10T1/2 cells and by the inefficiency of the MH1 or linker domain to interfere with BMP-2-mediated chondrogenic differentiation.

Expression of the inhibitory Smad7 in early mouse development and upregulation during embryonic vasculogenesis.

SMAD proteins are downstream targets of serine/threonine kinase receptors of the transforming growth factor beta (TGF beta) superfamily. Ligands activating these receptors regulate cell growth, differentiation and development in many tissues of various organisms. In mammals eight different Smad genes are known, each with different roles in mediating signalling between plasma membrane and nucleus. Smad6 and Smad7 are inhibitors of TGF beta family signalling. They are both expressed in human adult vascular endothelial cells, particularly after these cells have been subjected to shear stress (Topper et al. [1997] Proc Natl Acad Sci USA 94:9314-9319). Here we show by reverse transcriptase polymerase chain reaction and in situ hybridization that Smad7 mRNA is highly expressed in the developing vascular system of the mouse embryo but is also detectable much earlier in preimplantation embryos and during gastrulation. We also demonstrate by transient transgenesis that overexpression of Smad7 in mouse zygotes inhibits development beyond the 2-cell stage. This confirms earlier conclusions of similar, but complementary, experiments using a dominant negative type II TGF beta receptor demonstrating that TGF beta signalling is required for normal preimplantation development.

XSIP1, a Xenopus zinc finger/homeodomain encoding gene highly expressed during early neural development.

We have isolated a Xenopus homologue of the zinc finger/homeodomain-containing transcriptional repressor Smad-interacting protein-1 (SIP1) from mouse. XSIP1 is activated at the early gastrula stage and transcription occurs throughout embryogenesis. At the beginning of gastrulation, XSIP1 is strongly expressed in prospective neurectoderm. At the neurula stage, XSIP1 is highly expressed within the neural plate but weakly in the dorsal midline. At later stages of development transcripts are detected primarily within the neural tube and neural crest. In the adult, XSIP1 expression is detected at variable levels in several organs.

Region-specific activation of the Xenopus brachyury promoter involves active repression in ectoderm and endoderm: a study using transgenic frog embryos.

Tissue specification in the early embryo requires the integration of spatial information at the promoters of developmentally important genes. Although several response elements for signalling pathways have been identified in Xenopus promoters, it is not yet understood what defines the sharp borders that restrict expression to a specific tissue. Here we use transgenic frog embryos to study the spatial and temporal regulation of the Xbra promoter. Deletion analysis and point mutations in putative transcription factor-binding sites identified two repressor modules, which exert their main effects at different stages during gastrulation. One module is defined by a bipartite binding site for a Smad-interacting protein (SIP1) of the deltaEF1 repressor family and acts to confine expression to the marginal zone early in gastrulation. The other module is defined by two homeodomain-binding sites and is responsible for repression in dorsal mesoderm and ectoderm at mid-gastrula stages. In addition, an upstream region of the promoter is necessary to repress expression in neural tissues later in development. Together, our results show that repression plays an important role in the restriction of Xbra expression to the mesoderm, and we suggest that similar mechanisms may be involved in the spatial regulation of other genes in early embryonic development.

TTRAP, a novel protein that associates with CD40, tumor necrosis factor (TNF) receptor-75 and TNF receptor-associated factors (TRAFs), and that inhibits nuclear factor-kappa B activation.

CD40 belongs to the tumor necrosis factor (TNF) receptor family. CD40 signaling involves the recruitment of TNF receptor-associated factors (TRAFs) to its cytoplasmic domain. We have identified a novel intracellular CD40-binding protein termed TRAF and TNF receptor-associated protein (TTRAP) that also interacts with TNF-R75 and CD30. The region of the CD40 cytoplasmic domain that is required for TTRAP association overlaps with the TRAF6 recognition motif. Association of TTRAP with CD40 increases profoundly in response to treatment of cells with CD40L. Interestingly, TTRAP also associates with TRAFs, with the highest affinity for TRAF6. In transfected cells, TTRAP inhibits in a dose-dependent manner the transcriptional activation of a nuclear factor-kappaB (NF-kappaB)-dependent reporter mediated by CD40, TNF-R75 or Phorbol 12-myristate 13-acetate (PMA) and to a lesser extent by TRAF2, TRAF6, TNF-alpha, or interleukin-1beta (IL-1beta). TTRAP does not affect stimulation of NF-kappaB induced by overexpression of the NF-kappaB-inducing kinase (NIK), the IkappaB kinase alpha (IKKalpha), or the NF-kappaB subunit P65/RelA, suggesting it acts upstream of the latter proteins. Our results indicate that we have isolated a novel regulatory factor that is involved in signal transduction by distinct members of the TNF receptor family.

Smad5 is essential for left-right asymmetry in mice.

Left-right (L-R) asymmetry of the vertebrate body plan is established from an originally morphologically symmetric embryo. Recent studies have implicated several TGF-beta family signaling proteins (i.e., nodal, lefty-1, lefty-2, activin receptor type IIB, and Smad2) in L-R axis determination in the mouse. However, the genetic pathways underlying L-R patterning are still unclear. Smad5 is a downstream component in the TGF-beta family signaling cascade, and lack of Smad5 results in embryonic lethality between E9.5 and E11.5. In this report, we demonstrate that Smad5 mutant embryos have defects in heart looping and embryonic turning which are the first signs of L-R asymmetry in mice. To gain more insights into the molecular basis of the laterality defects in the Smad5-deficient embryos, we examined the expression of lefty-1, lefty-2, nodal, and Pitx2 since the asymmetric expression of these genes always closely correlates with the direction of heart looping and embryonic turning. In the absence of Smad5, lefty-1 was expressed at very low or undetectable levels, while nodal, lefty-2, and Pitx2 were expressed bilaterally. These data suggest that Smad5 is upstream of lefty-1, nodal, and lefty-2, and as a consequence also of Pitx2, and Smad5 is essential for L-R axis determination.

Differentiation-dependent alternative splicing and expression of the extracellular matrix protein 1 gene in human keratinocytes.

The human extracellular matrix protein 1 (Ecm1) gene is located at chromosome band 1q21 close to the epidermal differentiation complex and is transcribed in two discrete mRNAs: a full length Ecm1a and a shorter, alternatively spliced, Ecm1b transcript, the expression of which is restricted to tonsils and skin. The chromosomal localization and the Ecm1b expression in skin prompted us to investigate the role of Ecm1 in keratinocyte differentiation. In this study, we provide evidence for the existence of a relationship between keratinocyte differentiation and expression of the Ecm1b transcript. Cultures of subconfluent undifferentiated normal human keratinocytes express only Ecm1a. Upon reaching confluence, the cells start to differentiate, as measured by keratin K10 mRNA expression. Concomitantly Ecm1b mRNA expression is induced, although expression of Ecm1a mRNA remains unchanged. In addition, treatment of undifferentiated normal human keratinocyte cells with 12-O-tetradecanoyl-phorbol-13-acetate strongly induces the expression of Ecm1b mRNA. Expression of Ecm1b can also be induced by coculturing normal human keratinocytes with lethally irradiated feeder cells and by a diffusible factor secreted by stromal cells. In adult human skin, Ecm1a mRNA is expressed throughout the epidermis with the strongest expression in the basal and first suprabasal cell layers, whereas expression of Ecm1b mRNA is predominantly found in spinous and granular cell layers. Immunohistochemically, Ecm1a expression is almost completely restricted to the basal cell layer, whereas Ecm1b is detected in the suprabasal layers. These results are strongly suggestive of a role for Ecm1b in terminal keratinocyte differentiation, which is also supported by the localization of the Ecm1 gene at 1q21. Refinement of its genomic localization, however, placed Ecm1 centromeric of the epidermal differentiation complex.