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The Estrogen-responsive B Box Protein

A NOVEL REGULATOR OF KERATINOCYTE DIFFERENTIATION*
Open AccessPublished:March 27, 2002DOI:https://doi.org/10.1074/jbc.M111233200
      Keratinocyte growth factor (KGF) regulates proliferation, differentiation, migration, and survival of different types of epithelial cells, including keratinocytes of the skin. To gain insight into the mechanisms underlying these multiple functions, we searched for KGF- regulated genes in keratinocytes. Using the differential display reverse transcriptase-PCR technology, we identified the gene encoding the estrogen-responsive B box protein (EBBP) which has as yet not been functionally characterized. The full-length murine and human EBBP cDNAs were cloned and fully sequenced. They were shown to encode 75-kDa proteins, which are mainly localized in the cytoplasm of keratinocytes in vitro and in vivo. In vivo, EBBP was found at high levels in the KGF- and epidermal growth factor-responsive basal keratinocytes of human skin, but the expression was down-regulated in the hyperthickened epithelium of skin wounds. Stable overexpression of EBBP in HaCaT keratinocytes did not affect the proliferation rate of the transfected cells, but enhanced the early differentiation process. These results suggest that the presence of EBBP in basal keratinocytes is important for the differentiation capacity of these cells, and that down-regulation of EBBP expression in a hyperproliferative epithelium is required to maintain the cells in a non-differentiated stage.
      The epidermis forms the protective cover of the body surface. It is a multilayered renewal tissue composed mainly of keratinocytes. Terminally differentiated and dead cells are continuously lost from its surface and replaced by cells originating from the basal layer. Under normal conditions only cells touching on the basement membrane are able to proliferate (
      • Fuchs E.
      ). Transient hyperproliferation of keratinocytes occurs at the wound margins after skin injury (
      • Martin P.
      ), and permanent, uncontrolled hyperproliferation, together with abnormal differentiation of keratinocytes is a characteristic feature of major human skin diseases, such as psoriasis and epidermal cancers (
      • Carter D.M.
      ,
      • Christophers E.
      • Krueger G.G.
      ). Therefore, the balance between proliferation and differentiation must be tightly regulated. Cell-cell and cell-matrix interactions, as well as soluble factors such as hormones and growth factors are involved in this process, but the intracellular effectors have only partially been identified.
      To identify novel regulators of keratinocyte proliferation and differentiation, we searched for genes that are regulated by keratinocyte growth factor (KGF).
      The abbreviations used are: KGF
      keratinocyte growth factor
      FGF
      fibroblast growth factor
      DDRT-PCR
      differential display RT-PCR
      FCS
      fetal calf serum
      EGF
      epidermal growth factor
      HA
      hemagglutinin
      RBCC
      RING finger-B box-coiled coil
      TRIM
      tripartite motif
      1The abbreviations used are: KGF
      keratinocyte growth factor
      FGF
      fibroblast growth factor
      DDRT-PCR
      differential display RT-PCR
      FCS
      fetal calf serum
      EGF
      epidermal growth factor
      HA
      hemagglutinin
      RBCC
      RING finger-B box-coiled coil
      TRIM
      tripartite motif
      KGF (fibroblast growth factor 7, FGF-7) is a member of the FGF family, which is predominantly expressed by mesenchymal cells, but not by epithelial cells (
      • Rubin J.S.
      • Osada D.P.
      • Chedid M.
      • Miki T.
      • Ron D.
      • Cheon G.
      • Taylor W.G.
      • Fortney E.
      • Sakata H.
      • Finch P.W.
      • LaRochelle W.J.
      ). It acts in a paracrine manner by binding to its receptor, a splice variant of FGF receptor 2 that is expressed by different types of epithelial cells, including keratinocytes of the skin (
      • Miki T.
      • Bottaro D.P.
      • Fleming T.P.
      • Smith C.L.
      • Burgess W.H.
      • Chan A.M.
      • Aaronson S.A.
      ). This ligand-receptor interaction has been implicated in morphogenetic processes of epithelial tissues and in cell survival under stress conditions (
      • Werner S.
      ). KGF expression is strongly induced upon injury to various epithelial tissues in normal and pathological situations (
      • Werner S.
      ,
      • Werner S.
      • Peters K.G.
      • Longaker M.T.
      • Fuller-Pace F.
      • Banda M.
      • Williams L.T.
      ,
      • Marchese C.
      • Chedid M.
      • Dirsch O.R.
      • Csaky K.G.
      • Santanelli F.
      • Latini C.
      • LaRochelle W.J.
      • Torrisi M.R.
      • Aaronson S.A.
      ), and activation of the KGF receptor was shown to be important for efficient re-epithelialization of skin wounds (
      • Werner S.
      • Smola H.
      • Liao X.
      • Longaker M.T.
      • Krieg T.
      • Hofschneider P.H.
      • Williams L.T.
      ).
      In vitro, KGF stimulates keratinocyte migration and proliferation (
      • Tsuboi R.
      • Sato C.
      • Kurita Y.
      • Ron D.
      • Rubin J.S.
      • Ogawa H.
      ,
      • Rubin J.S.
      • Osada H.
      • Finch P.W.
      • Taylor W.G.
      • Rudikoff S.
      • Aaronson S.A.
      ), and it inhibits terminal differentiation and apoptosis of these cells (
      • Hines M.D.
      • Allen-Hoffmann B.L.
      ). Nevertheless, it also induces the expression of differentiation–specific proteins in response to an increase in the extracellular Ca2+ concentration, and it has been suggested that KGF plays a crucial role in the initiation of the early differentiation program (
      • Marchese C.
      • Rubin J.
      • Ron D.
      • Faggioni A.
      • Torrisi M.R.
      • Messina A.
      • Frati L.
      • Aaronson S.A.
      ,
      • Marchese C.
      • Sorice M., De
      • Stefano C.
      • Frati L.
      • Torrisi M.R.
      ).
      To gain insight into the changes in gene expression after binding of KGF to its receptor, we performed differential display RT-PCR (DDRT-PCR) with RNAs from quiescent and KGF-stimulated HaCaT keratinocytes. By this method we identified the ebbp gene as a target of KGF action. The latter was originally identified as a gene, which is regulated by estrogen and tamoxifen in human mammary epithelial cells stably expressing an estrogen receptor mutant (
      • Liu H.-L. C.
      • Golder-Novoselsky E.
      • Seto M.H.
      • Webster L.
      • McClary J.
      • Zajchowski D.A.
      ). It is ubiquitously expressed in human tissues, but the function of the encoded protein is completely unknown. In the present study, we have characterized the EBBP protein, and we demonstrate a novel role of EBBP in the regulation of keratinocyte differentiation.

      DISCUSSION

      In this study we describe a novel KGF-regulated gene which encodes the EBBP protein. The ebbp gene has recently been described as a gene that is regulated by estrogen in mammary epithelial cells stably transfected with an estrogen receptor cDNA (
      • Liu H.-L. C.
      • Golder-Novoselsky E.
      • Seto M.H.
      • Webster L.
      • McClary J.
      • Zajchowski D.A.
      ). It is ubiquitously expressed in various human tissues (
      • Liu H.-L. C.
      • Golder-Novoselsky E.
      • Seto M.H.
      • Webster L.
      • McClary J.
      • Zajchowski D.A.
      ). However, nothing is as yet known about the function of the encoded protein.
      EBBP contains several interesting domains (Figs. 2 and 3 A). An acidic domain at the amino terminus is followed by two B boxes and three α-helices which may form a coiled-coil domain. Two motifs within these α-helices, F XXLL and L XXLL, have been identified as nuclear receptor interaction domains in transcriptional co-factors such as TIF1, NSD-1, and CBP (
      • Heery D.M.
      • Kalkhoven E.
      • Hoare S.
      • Parker M.G.
      ,
      • Huang N.
      • vom Baur E.
      • Garnier J.-M.
      • Lerouge T.
      • Vonesch J.-L.
      • Lutz Y.
      • Chambon P.
      • Losson R.
      ). At the carboxyl terminus, an RFP or B30.2 domain was detected. This domain composition is characteristic of a class of proteins called RING B-box coiled-coil (RBCC) proteins (
      • Borden K.L.B.
      ) or tripartitemotif (TRIM) proteins (
      • Reymond A.
      • Meroni G.
      • Fantozzi A.
      • Merla G.
      • Cairo S.
      • Luzi L.
      • Riganelli D.
      • Zanaria E.
      • Messali S.
      • Cainarca S.
      • Guffanti A.
      • Minucci S.
      • Pelicci P.G.
      • Ballabio A.
      ). Although EBBP does not contain a RING finger, the other domains are present in this protein. RING fingers and B boxes are cysteine- and histidine-rich zinc-binding motifs mediating protein-protein interactions. The B box is important for homo- and heterodimerization of the RBCC proteins (
      • Reymond A.
      • Meroni G.
      • Fantozzi A.
      • Merla G.
      • Cairo S.
      • Luzi L.
      • Riganelli D.
      • Zanaria E.
      • Messali S.
      • Cainarca S.
      • Guffanti A.
      • Minucci S.
      • Pelicci P.G.
      • Ballabio A.
      ,
      • Borden K.L.B.
      ).
      The biological functions of RBCC proteins have been only partially characterized. Most of them are involved in the regulation of cell growth and differentiation during development and in the adult organism. Most interestingly, mutations in RBCC and other B box containing proteins have been found in several human diseases. For example, a translocation generating a fusion protein between the PML gene product and the retinoic acid receptor α is associated with promyelocytic leukemia, and the transcriptional co-activator TIF1 was found to be fused to the tyrosine kinase RET in childhood thyroid carcinomas. Mutations in the B30.2 domain of pyrin have been found in familial Mediterranean fever, and mutations in the B30.2 domain of midin cause Opitz syndrome, a multiple congenital anomaly manifested by abnormal closure of midline structures (
      • Torok M.
      • Etkin L.D.
      ). These findings suggest that RING finger and B box proteins regulate crucial cellular functions.
      In this study we have characterized the EBBP protein and we studied its regulation in keratinocytes. We demonstrate that the human EBBP protein present in COS-1 cells and HaCaT cells has an apparent molecular weight of 75,000, a finding which is consistent with previous data obtained with HepG2 cells (
      • Liu H.-L. C.
      • Golder-Novoselsky E.
      • Seto M.H.
      • Webster L.
      • McClary J.
      • Zajchowski D.A.
      ). Since the calculated molecular weight is only 64,000, it seems likely that the protein is post-translationally modified. This hypothesis is supported by the presence of several putative phosphorylation sites in the protein, and by our observation that both endogenous EBBP and EBBP-HA often run as double bands in SDS gels.
      In the skin, EBBP was strongly expressed in the proliferation-competent, non-differentiated keratinocytes of the basal layer. These cells are also targets of KGF and EGF action (
      • Werner S.
      • Peters K.G.
      • Longaker M.T.
      • Fuller-Pace F.
      • Banda M.
      • Williams L.T.
      ,
      • King L.E., Jr.
      • Gates R.E.
      • Stoscheck C.M.
      • Nanney L.B.
      ), indicating that the maintenance of high levels of EBBP by KGF and EGF observed in vitro might also be important in normal skin in vivo. However, despite the high levels of KGF and EGF receptor ligands in skin wounds (
      • Werner S.
      • Peters K.G.
      • Longaker M.T.
      • Fuller-Pace F.
      • Banda M.
      • Williams L.T.
      ,
      • Marchese C.
      • Chedid M.
      • Dirsch O.R.
      • Csaky K.G.
      • Santanelli F.
      • Latini C.
      • LaRochelle W.J.
      • Torrisi M.R.
      • Aaronson S.A.
      ,
      • Todd R.
      • Donoff B.R.
      • Chiang T.
      • Chou M.Y.
      • Elovic A.
      • Gallagher G.T.
      • Wong D.T.
      ,
      • Marikowsky M.
      • Breuing K.
      • Liu P.Y.
      • Eriksson E.
      • Higashiyama S.
      • Farber P.
      • Abraham J.
      • Klagsbrun M.
      ), expression of EBBP was strongly down-regulated in the hyperthickened wound epithelium, indicating the presence of other, as yet unidentified factors in a wound that suppress ebbp gene expression.
      The hyperproliferative wound epithelium is characterized by the presence of rapidly proliferating keratinocytes with reduced differentiation capacity (
      • Martin P.
      ). The significantly reduced levels of EBBP seen in these cells suggest a role of EBBP in the regulation of keratinocyte proliferation and/or differentiation. This hypothesis is supported by preliminary results from our laboratory demonstrating that EBBP levels are also reduced in the hyperthickened epidermis of psoriatic patients, and particularly in the keratinocytes of basal cell carcinomas. Most importantly, the results obtained with our HaCaT cells that overexpress EBBP provide functional evidence for this hypothesis. Whereas proliferation was not altered in these cells, EBBP overexpression strongly stimulated the early differentiation process. This finding suggests that the presence of high levels of EBBP in keratinocytes is important for the onset of keratinocyte differentiation under permissive conditions. Thus, EBBP could play an important role in the induction of the differentiation pathway, a finding that is consistent with the common roles of RBCC members and related proteins in the regulation of cell growth and differentiation during development and in the adult organism. In addition to a possible role of EBBP in early differentiation, the strong EBBP staining of the cornified layer indicates an additional role of EBBP in late differentiation. In a hyperproliferative epithelium as seen in wounded and psoriatic skin and in basal cell carcinomas, differentiation is inhibited, and down-regulation of ebbp expression might be an important prerequisite for maintaining the cells in a non-differentiated stage.
      The molecular mechanisms of EBBP action are as yet unknown. However, the presence of several protein domains that are characteristic for transcriptional regulators, suggests that EBBP can directly or indirectly modulate gene transcription. This hypothesis is supported by the recently observed DNA-binding capacity of EBBP in the presence of all cellular proteins.
      H.-D. Beer, unpublished data.
      The presence of two B boxes in conjunction with the coiled-coil domain in the EBBP protein suggests that it acts as a homo- or heterodimer or -multimer as shown for other TRIM family members (
      • Reymond A.
      • Meroni G.
      • Fantozzi A.
      • Merla G.
      • Cairo S.
      • Luzi L.
      • Riganelli D.
      • Zanaria E.
      • Messali S.
      • Cainarca S.
      • Guffanti A.
      • Minucci S.
      • Pelicci P.G.
      • Ballabio A.
      ). Indeed, preliminary co-precipitation experiments revealed that EBBP does indeed form homomultimers.
      C. Munding, unpublished data.
      In addition, EBBP is likely to interact with other, as yet unidentified proteins, as suggested by the presence of several protein-protein interaction domains. In particular, the L XXLL domains are responsible for interaction with nuclear receptors such as the retinoic acid receptors (
      • Heery D.M.
      • Kalkhoven E.
      • Hoare S.
      • Parker M.G.
      ,
      • Huang N.
      • vom Baur E.
      • Garnier J.-M.
      • Lerouge T.
      • Vonesch J.-L.
      • Lutz Y.
      • Chambon P.
      • Losson R.
      ). These interacting proteins could also be responsible for the observed DNA-binding capacity, since EBBP lacks a DNA-binding domain. The predominant cytoplasmic localization of EBBP suggests that it retains possible DNA-binding interaction partners in this compartment, thereby inhibiting their function.
      In summary, we have identified EBBP as the product of a novel KGF-regulated gene. Its expression in the KGF-responsive keratinocytes of normal skin as well as the induction of the differentiation program in EBBP-transfected HaCaT cells suggests that it mediates, at least in part, the differentiation stimulating activity of KGF under differentiation-permissive conditions (
      • Marchese C.
      • Rubin J.
      • Ron D.
      • Faggioni A.
      • Torrisi M.R.
      • Messina A.
      • Frati L.
      • Aaronson S.A.
      ,
      • Marchese C.
      • Sorice M., De
      • Stefano C.
      • Frati L.
      • Torrisi M.R.
      ). Since EBBP is also expressed at high levels in various other epithelial cells,2 it will be important to determine whether it also affects the balance between proliferation and differentiation of these cell types in vitro, as well in development, repair, and/or disease.

      ACKNOWLEDGEMENTS

      We thank Andreas Stanzel and Christiane Born-Berclaz for excellent technical assistance, Dr. Cornelia Mauch for the human skin biopsies, and Dr. P. Boukamp for providing HaCaT keratinocytes.

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