Bone morphogenetic protein-2 (BMP-2) transactivates Dlx3 through Smad1 and Smad4: alternative mode for Dlx3 induction in mouse keratinocytes.

Expression of the Dlx3 homeodomain gene is induced in terminally differentiated epidermal cells. Dlx3 regulates gene expression in skin and plays important roles in patterning of the embryonic ectoderm through differential sensitivity to bone morphogenetic protein (BMP) signaling. We analyzed the expression of BMP family members in murine keratinocytes; BMP-2 is expressed in proliferative basal and differentiated suprabasal keratinocytes. BMP-2 induced transcription of Dlx3 within 12 h of treatment of keratinocytes cultured in vitro. We proceeded to delineate the BMP-2-responsive region to an area between -1917 and -1747 in the Dlx3 promoter. Gel shift assays with recombinant Smad1 and Smad4 demonstrated that this DNA fragment (-1917 to -1747) was competent in the formation of protein-DNA complexes. By deletion and mutational analyses we localized a Smad1/Smad4-binding site containing a GCAT motif, which showed similarity to other TGF-beta family responsive elements. Supershift assays with keratinocyte nuclear extracts and antibodies against members of the Smad family showed that this motif was able to form a complex with Smad1. Mutation of the Smad1/Smad4-binding site inhibited transcriptional activation of the Dlx3 gene by BMP-2. In the hair follicle, where Dlx3 is expressed in the hair matrix cells, BMP-2 also activates Dlx3 transcription. These results provide a possible mechanism of action for the BMP signaling pathway on the regulation of Dlx3.

[1]  K. Kinzler,et al.  Human Smad3 and Smad4 are sequence-specific transcription activators. , 1998, Molecular cell.

[2]  Yigong Shi,et al.  Crystal Structure of a Smad MH1 Domain Bound to DNA Insights on DNA Binding in TGF-β Signaling , 1998, Cell.

[3]  L. Attisano,et al.  The Smads , 2001, Genome Biology.

[4]  M. Beanan,et al.  Regulation and function of Dlx3 in vertebrate development , 2000, Developmental dynamics : an official publication of the American Association of Anatomists.

[5]  J. Lim,et al.  Differential regulation of Dlx gene expression by a BMP morphogenetic gradient. , 2001, The International journal of developmental biology.

[6]  B Geronimo,et al.  Targeted misexpression of constitutively active BMP receptor-IB causes bifurcation, duplication, and posterior transformation of digit in mouse limb. , 2000, Developmental biology.

[7]  B. Hogan,et al.  Transgenic mice as a model to study the role of TGF-beta-related molecules in hair follicles. , 1993, Genes & development.

[8]  P M Steinert,et al.  Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro , 1989, The Journal of cell biology.

[9]  H. Shibuya,et al.  A BMP-inducible gene, dlx5, regulates osteoblast differentiation and mesoderm induction. , 1999, Developmental biology.

[10]  K. Struhl,et al.  Current Protocols in Molecular Biology (New York: Greene Publishing Associates and Wiley-Interscience). Host-Range Shuttle System for Gene Insertion into the Chromosomes of Gram-negative Bacteria. , 1988 .

[11]  W. Knöchel,et al.  Smad1 and Smad4 Are Components of the Bone Morphogenetic Protein-4 (BMP-4)-induced Transcription Complex of the Xvent-2B Promoter* , 2000, The Journal of Biological Chemistry.

[12]  K. Kinzler,et al.  Characterization of human FAST-1, a TGFβ and activin signal transducer , 1998 .

[13]  M. Beanan,et al.  Inhibitory patterning of the anterior neural plate in Xenopus by homeodomain factors Dlx3 and Msx1. , 1999, Developmental biology.

[14]  B. Hogan,et al.  Patterns of expression of murine Vgr-1 and BMP-2a RNA suggest that transforming growth factor-beta-like genes coordinately regulate aspects of embryonic development. , 1989, Genes & development.

[15]  M. Morasso,et al.  Regulation of the Dlx3 Homeobox Gene upon Differentiation of Mouse Keratinocytes* , 1999, The Journal of Biological Chemistry.

[16]  G. Robinson,et al.  Differential and overlapping expression domains of Dlx-2 and Dlx-3 suggest distinct roles for Distal-less homeobox genes in craniofacial development , 1994, Mechanisms of Development.

[17]  S. Yuspa,et al.  Coordinate changes in gene expression which mark the spinous to granular cell transition in epidermis are regulated by protein kinase C , 1993, The Journal of cell biology.

[18]  C. Heldin,et al.  Identification and Functional Characterization of a Smad Binding Element (SBE) in the JunB Promoter That Acts as a Transforming Growth Factor-β, Activin, and Bone Morphogenetic Protein-inducible Enhancer* , 1998, The Journal of Biological Chemistry.

[19]  R. Derynck,et al.  Smad3 and Smad4 cooperate with c-Jun/c-Fos to mediate TGF-β-induced transcription , 1998, Nature.

[20]  M. Morasso,et al.  Phosphorylation of murine homeodomain protein Dlx3 by protein kinase C , 2001, FEBS letters.

[21]  M. Morasso,et al.  The homeodomain gene Xenopus Distal-less-like-2 (Xdll-2) is regulated by a conserved mechanism in amphibian and mammalian epidermis. , 1994, Developmental biology.

[22]  K. Miyazono,et al.  Synergistic signaling in fetal brain by STAT3-Smad1 complex bridged by p300. , 1999, Science.

[23]  N. Andrews,et al.  A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. , 1991, Nucleic acids research.

[24]  P. Hoodless,et al.  Smad2 and Smad3 positively and negatively regulate TGF beta-dependent transcription through the forkhead DNA-binding protein FAST2. , 1998, Molecular cell.

[25]  K. Miyazono,et al.  Characterization of a bone morphogenetic protein-responsive Smad-binding element. , 2000, Molecular biology of the cell.

[26]  B. Hogan,et al.  Bone morphogenetic proteins: multifunctional regulators of vertebrate development. , 1996, Genes & development.

[27]  A. Reddi,et al.  Bone morphogenetic proteins: an unconventional approach to isolation of first mammalian morphogens. , 1997, Cytokine & growth factor reviews.

[28]  D. Bowden,et al.  Identification of a mutation in DLX3 associated with tricho-dento-osseous (TDO) syndrome. , 1998, Human molecular genetics.

[29]  E. Fuchs,et al.  The epidermis: rising to the surface. , 1994, Current opinion in genetics & development.

[30]  M. Morasso,et al.  Transcriptional activation by the homeodomain protein distal-less 3. , 1999, Nucleic acids research.

[31]  X. Cao,et al.  Smad1 Interacts with Homeobox DNA-binding Proteins in Bone Morphogenetic Protein Signaling* , 1999, The Journal of Biological Chemistry.

[32]  X. Cao,et al.  Smad1 Domains Interacting with Hoxc-8 Induce Osteoblast Differentiation* , 2000, The Journal of Biological Chemistry.

[33]  M. Morasso,et al.  A Xenopus distal-less gene in transgenic mice: conserved regulation in distal limb epidermis and other sites of epithelial-mesenchymal interaction. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[34]  Kirby D. Johnson,et al.  Drosophila Mad binds to DNA and directly mediates activation of vestigial by Decapentaplegic , 1997, Nature.

[35]  Xin Chen,et al.  A transcriptional partner for MAD proteins in TGF-β signalling , 1996, Nature.

[36]  R. Mcinnes,et al.  The Tlx-2 homeobox gene is a downstream target of BMP signalling and is required for mouse mesoderm development. , 1998, Development.

[37]  Denis Vivien,et al.  Direct binding of Smad3 and Smad4 to critical TGFβ‐inducible elements in the promoter of human plasminogen activator inhibitor‐type 1 gene , 1998, The EMBO journal.

[38]  W. Pledger,et al.  Expression and growth inhibitory effect of decapentaplegic Vg-related protein 6: evidence for a regulatory role in keratinocyte differentiation. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[39]  J. Wrana,et al.  Smads as transcriptional co-modulators. , 2000, Current opinion in cell biology.

[40]  M. Morasso,et al.  Regulation of epidermal differentiation by a Distal-less homeodomain gene , 1996, The Journal of cell biology.

[41]  K. Miyazono,et al.  Signal transduction by bone morphogenetic proteins. , 1998, Cytokine & growth factor reviews.