J Inv Der-Liu-comment Widelitz

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See related article on page 264RegulatingtheRegulators:RoutingtheWnt-b-Catenin–LefSignalsRandall B. WidelitzDepartment of Pathology, University of Southern California, Keck School of Medicine, Los Angeles, CaliforniaAs genomic sequences are identified, the next frontier to- staining the mesenchyme (E11) and epithelium (by E12) ofward effective genetic medicine will be to identify how gene wild-type mouse vibrissae. Constant expression in the me-expressionisregulated,soectopicgenescanbeexpressed senchyme but transient expression of Lef-1 in the epithe-in appropriate locations, at appropriate times and to ap- lium is required for normal whisker formation. Lef-1 knock-propriate therapeutic levels. This is especially true for mol- out mice lack hairs, teeth, mammary glands, and other ep-ecules that regulate the downstream expression of a ithelial-derived skin appendages (van Genderen et al, 1994).cascade of genes. Like traffic control regulators, these Mice expressing an NH -terminal truncation of Lef-1 in the2molecules effectively act as molecular switches, which can skin epidermis form interfollicular skin rather than hairs orfavor one pathway or another with dramatic biological con- formsebaceoustumors(Niemannetal,2002).b-Catenin andsequences. For years scientists have tried to map the Lef-1 form a complex to suppress E-cadherin expressiontranscriptional regulatory regions of structural effector presumably while inducing the expression of other genesmolecules ...

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See related article on page 264

Regulating the Regulators: Routing the Wnt-b-Catenin–Lef Signals

Randall B. Widelitz Department of Pathology, University of Southern California, Keck School of Medicine, Los Angeles, California

As genomic sequences are identified, the next frontier to- ward effective genetic medicine will be to identify how gene expression is regulated, so ectopic genes can be expressed in appropriate locations, at appropriate times and to ap- propriate therapeutic levels. This is especially true for mol- ecules that regulate the downstream expression of a cascade of genes. Like traffic control regulators, these molecules effectively act as molecular switches, which can favor one pathway or another with dramatic biological con- sequences. For years scientists have tried to map the transcriptional regulatory regions of structural effector molecules and found that gene promoters are composed of multiple small, positive, and negative gene response el- ements. But the study of the regulatory regions of signaling molecules has lagged. Gene regulation is based on a ‘‘committee level decision’’ of these transcriptional factors and co-activators. In recent years, several key signaling pathways have been shown to play major roles in regulation of skin/skin appendage morphogenesis (e.g., Wnt,b-cat- enin, Shh, FGF, BMP). The regulation of these regulators has tremendous consequences. Here we will comment on Wnt signaling based upon new progress made on the lymph- ocyte enhancer factor (Lef)-1 promoter by Liuet al(2004).

Recent Progress in Wnt Signaling

Wnt signaling through the canonical pathway stabilizesb- catenin and allows it to translocate to the nucleus, asso- ciate with Lef/T cell factor (Tcf) and induce transcriptional events. The Wnt-b-catenin signaling cascade has been shown to be important for developmental morphogenesis and cancer. It plays an essential role in the morphogenesis of several organs including skin appendages. Over expres- sion of exogenous, constitutively activeb-catenin in other- wise normal mice led to the formation of ectopic hairs and the formation of hair tumors (Gatet al, 1998). Cre/loxP knockout mice demonstrated thatb-catenin plays a critical role in directing epidermal cell fate toward becoming hair follicle or interplacode epidermis (Huelskenet al, 1991). Similarly, inhibition of Wnt signaling with Dkk-1 blocked the initiation of hair follicle development and caused other de- velopmental anomalies (Andlet al, 2002). Althoughb-cat- enin is an important component in these events, it does not act alone to induce hair formation. It must interact with co- activators. Lef-1 mRNA and protein have been found in the developing hair. Lef-1 protein was detected by immuno-

Abbreviations: Lef, lymphocyte enhancer factor; Tcf, T cell factor

staining the mesenchyme (E11) and epithelium (by E12) of wild-type mouse vibrissae. Constant expression in the me- senchyme but transient expression of Lef-1 in the epithe- lium is required for normal whisker formation. Lef-1 knock- out mice lack hairs, teeth, mammary glands, and other ep- ithelial-derived skin appendages (van Genderenet al, 1994). Mice expressing an NH 2 -terminal truncation of Lef-1 in the skin epidermis form interfollicular skin rather than hairs or form sebaceous tumors (Niemannet al, 2002).b-Catenin and Lef-1 form a complex to suppress E-cadherin expression presumably while inducing the expression of other genes within the hair follicle (Jamoraet al, 2003). These studies clearly show the importance of the canonical Wnt signaling pathway and of Lef-1 to skin appendage formation. b-Catenin can interact with a number of downstream genes to activate different pathways. The different Lef/Tcf family members can form a complex withb-catenin to in- duce a different set of downstream genes, perhaps by at- tracting different transcriptional co-activators. For example, Lef-1 can induceSiamois, and Tcf-4E can induce the cau- dal-related homeobox gene,Cdx1(Hecht and Stemmler, 2003). Tcf-1 is essential for thymocyte differentiation (Verbeeket al, 1995), Tcf-3 is more involved in axis dupli- cation (Merrillet al, 2001), Tcf-4 regulates the formation of intestinal epithelium (Korineket al, 1998), and Lef-1 affects epithelial-mesenchymal interactions in many organ systems (van Genderenet al, 1994). In the skin,b-catenin interacting with Lef-1 leads to hair formation, but interactions with TCF- 3 lead to sebaceous gland formation (Merrillet al, 2001). Additionally,b-catenin has been shown to directly inter- act with a number of other nuclear transcriptional repressor and co-activator proteins including XSox17, Xsox3, Smad proteins, the retinoic acid receptor, and androgen receptor leading to the transcriptional activation of other genes.b- Catenin association with either of the steroid receptors re- duces its association with Lef/Tcf and thereby inhibits Lef/ Tcf mediated transcription. Thus, there is competition for binding tob-catenin, which may further regulate the fa- cilitative recruitment of other co-activators to stimulate or repress the transcription of different cohorts of downstream genes. In this way, the interaction ofb-catenin with its varied partners can have dramatic consequences on the activation of downstream effector pathways. A cell’s predilection to- ward one pathwayversusanother is dependent to a certain extent on the differential expression of these regulatory molecules within the cellular milieu (Fig 1). Signaling through the Wnt pathway has been implicated in tumor formation in a number of different tissues. In the skin over expression ofb-catenin led to the induction of hair

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REGULATING THE REGULATORSix

In this issue, Liuet alhave further examined regulation of the Lef-1 promoter in the context of chromatin structure which places more constraints on gene expressionin vivo. Using transgenic mice with LacZ expression driven by dif- ferent forms of the Lef-1 promoter, they report that the 2.5 kb human promoter region is sufficient to direct expression within hairs and vibrissae, although the timing of expression was delayed compared to earlier reports based on immuno- staining. Expression from the transgene was only observed in the mesenchyme, not in the epithelium in contrast to these earlier reports. A longer construct, containing3 kb downstream of the initiation codon (including intron 1 and a portion of intron 2) in addition to the 2.5 kb promoter, di- rected expression to the sebaceous glands. This expres- sion, although not previously described, was confirmed in wild-type embryos. This demonstrates that intronic regula- tory sequences further restrict expression. Transgenic ex- pression of a 2.5 kb construct lacking the WRE failed to express in the mesenchyme, raising the likelihood that Wnt function directs mesenchymal expression of Lef-1. Further- more, expression in one of the transgenic mice mimicked Figure 1 the normal epithelial pattern. Hence, activation or repres- Coactivator competition forbcatenin can route cell fate.b-Cat- sion of the 110 bp WRE may direct expression to either the enin can directly interact with several competing transcriptional co- mesenchyme or epithelium. They then go on to show that activators which preferentially transcribe different downstream genes. Shh can induceb-galactosidase expression from the 2.5 kb Here this is depicted as a traffic circle with pathways diverging in dif- ferent directions based on the co-activator associated withb-catenin. Lef-1 promoter as is normally seen in wild-type mice after In this scenario, the relative levels of expression of these important Shh induces telogen phase hairs to enter anagen phase. regulatory molecules can route cells towards alternative fates. There- Discrepancies between endogenous and transgene expres- fore it is important to study how these regulators are themselves regulated. sion suggest that some elements of the Lef-1 promoter are missing in their tested constructs or may be configured dif- ferently than in their normal state. tumors (Gatet al, 1998). Mutations ofb-catenin have alsoStudies of this type will begin to elucidate positive and been found in naturally occurring hair tumors (Chanet alregulatory elements controlling the expression of, negative 1999). One might envision that differential expression of the signaling molecules. Cellular conditions can favor the acti- b-catenin partner molecules would influence tumor forma-vation of one pathway or another. If the expression of these tion? This again highlights the importance of understandingLef/Tcf factors is transcriptionally regulated,b-catenin will the regulation ofb preferentially bind to another partner protein, ultimately -catenin and its associated proteins. changing cell fate. Given the region-specific, hormone-de- pendent sexual dimorphism of skin appendages in humans and other species (i.e., beards grow on males but not fe- males; male pattern baldness, etc.), this is an area that Regulation of Wnt Signaling Regulators warrants further exploration. Once this information is as- Much attention has been focused on the role of nuclearcertained, it can be utilized for modulating the course of translocation ofb-catenin in activating the canonical path- disease. Therefore, it is essential that we understand how way. Regulation, however, also takes place at the transcrip-these regulatory molecules are themselves regulated at the tional level. We found in chicken skin thatbtranslational, and post-translational levels.-catenin transcriptional, transcripts were dynamically regulated (Widelitzet al, DOI: 10.1111/j.0022-202X.2004.23239.x 2000). In the rat, mouse, and human, Liet al(2004) have examined theb-catenin promoter and found many regula- Author correspondence to: Randall Widelitz, PhD, Department of Pa- tory elements by gel shift, reporter assays on serial deletion thology, University of Southern California, Keck School of Medicine, constructsin vitroand protein/DNA analyses. The function HMR 305D, 2011 Zonal Avenue, Los Angeles, CA 90033. Email: wide- litz@pathfinder.hsc.usc.edu of some regions of the promoter region had been previously testedin vitro(Nolletet al, 1996). Similar studies have been performed on the Lef-1 promoter (Hovaneset al, 2000; Filali References et al, 2002). Filaliet al(2002) found that a2.5 kb region of the human promoter was inducible by Wnt-3a andb-cat- Andl T, Reddy ST, Gaddapara T, Millar SE: Wnt signals are required for the in- enin. Some regions of the promoter led to increased ex- itiation of hair follicle development. Dev Cell 2:643–653, 2002 Chan EF, Gat U, McNiff JM, Fuchs E: A common human skin tumour is caused by pression while others suppressed expression. They then activating mutations in beta-catenin. Nat Genet 21:410–413, 1999 went on to identify a 110 bp Wnt responsive element (WRE) Filali M, Cheng N, Abbott D, Leontiev V, Engelhardt JF: Wnt-3A/b-catenin sig- within the Lef-1 promoter. Wnt3a relieved inhibition from this naling induces transcription from the Lef-1 promoter. J Biol Chem 277: site and promoted Lef-1 transcription. 33398–33410, 2002

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THE JOURNALOF INVESTIGATIVE DERMATOLOGY

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