FGF4, a direct target of LEF1 and Wnt signaling, can rescue the arrest of tooth organogenesis in Lef1(−/−) mice

Lymphoid enhancer factor (LEF1), a nuclear mediator of Wnt signaling, is required for the formation of organs that depend on inductive interactions between epithelial and mesenchymal tissues. In previous tissue recombination experiments with normal and Lef1(−/−) tooth germs, we found that the effect of LEF1 expression in the epithelium is tissue nonautonomous and transferred to the subjacent mesenchyme. Here we examine the molecular basis for LEF1 function and find that the epithelium of the developmentally arrested Lef1(−/−) tooth rudiments fails to express Fgf4, Shh, and Bmp4, but not Wnt10a. We identify the Fgf4 gene as a direct transcriptional target for LEF1 and show that beads soaked with recombinant FGF4 protein can fully overcome the developmental arrest of Lef1(−/−) tooth germs. In addition, we find that FGF4 beads induce rapidly the expression of Fgf3 in dental mesenchyme and that both epithelial and mesenchymal FGF proteins induce the delayed expression of Shh in the epithelium. Taken together, these data indicate that a single target of LEF1 can account for the function of LEF1 in tooth development and for a relay of a Wnt signal reception to a cascade of FGF signaling activities, allowing for a sequential and reciprocal communication between epithelium and mesenchyme

Recruitment of RNA polymerase II cofactor PC4 to DNA damage sites.

The multifunctional nuclear protein positive cofactor 4 (PC4) is involved in various cellular processes including transcription, replication, and chromatin organization. Recently, PC4 has been identified as a suppressor of oxidative mutagenesis in Escherichia coli and Saccharomyces cerevisiae. To investigate a potential role of PC4 in mammalian DNA repair, we used a combination of live cell microscopy, microirradiation, and fluorescence recovery after photobleaching analysis. We found a clear accumulation of endogenous PC4 at DNA damage sites introduced by either chemical agents or laser microirradiation. Using fluorescent fusion proteins and specific mutants, we demonstrated that the rapid recruitment of PC4 to laser-induced DNA damage sites is independent of poly(ADP-ribosyl)ation and gammaH2AX but depends on its single strand binding capacity. Furthermore, PC4 showed a high turnover at DNA damages sites compared with the repair factors replication protein A and proliferating cell nuclear antigen. We propose that PC4 plays a role in the early response to DNA damage by recognizing single-stranded DNA and may thus initiate or facilitate the subsequent steps of DNA repair

Keratins Mediate Localization of Hemidesmosomes and Repress Cell Motility

The keratin (K)–hemidesmosome (HD) interaction is crucial for cell-matrix adhesion and migration in several epithelia, including the epidermis. Mutations in constituent proteins cause severe blistering skin disorders by disrupting the adhesion complex. Despite extensive studies, the role of keratins in HD assembly and maintenance is only partially understood. Here we address this issue in keratinocytes in which all keratins are depleted by genome engineering. Unexpectedly, such keratinocytes maintain many characteristics of their normal counterparts. However, the absence of the entire keratin cytoskeleton leads to loss of plectin from the hemidesmosomal plaque and scattering of the HD transmembrane core along the basement membrane zone. To investigate the functional consequences, we performed migration and adhesion assays. These revealed that, in the absence of keratins, keratinocytes adhere much faster to extracellular matrix substrates and migrate approximately two times faster compared with wild-type cells. Reexpression of the single keratin pair K5 and K14 fully reversed the above phenotype. Our data uncover a role of keratins, which to our knowledge is previously unreported, in the maintenance of HDs upstream of plectin, with implications for epidermal homeostasis and pathogenesis. They support the view that the downregulation of keratins observed during epithelial–mesenchymal transition supports the migratory and invasive behavior of tumor cells

Skin Fragility and Impaired Desmosomal Adhesion in Mice Lacking All Keratins

Keratins perform major structural and regulatory functions in epithelia. Owing to redundancy, their respective contribution to epidermal integrity, adhesion, and cell junction formation has not been addressed in full. Unexpectedly, the constitutive deletion of type II keratins in mice was embryonic lethal ∼E9.5 without extensive tissue damage. This prompted us to analyze keratin functions in skin where keratins are best characterized. Here, we compare the mosaic and complete deletion of all type II keratins in mouse skin, with distinct consequences on epidermal integrity, adhesion, and organismal survival. Mosaic knockout (KO) mice survived ∼12 days while global KO mice died perinatally because of extensive epidermal damage. Coinciding with absence of keratins, epidermal fragility, inflammation, increased epidermal thickness, and increased proliferation were noted in both strains of mice, accompanied by significantly smaller desmosomes. Decreased desmosome size was due to accumulation of desmosomal proteins in the cytoplasm, causing intercellular adhesion defects resulting in intercellular splits. Mixing different ratios of wild-type and KO keratinocytes revealed that ∼60% of keratin-expressing cells were sufficient to maintain epithelial sheets under stress. Our data reveal a major contribution of keratins to the maintenance of desmosomal adhesion and epidermal integrity with relevance for the treatment of epidermolysis bullosa simplex and other keratinopathies

New Roles for Glial Cell Line-Derived Neurotrophic Factor and Neurturin : Involvement in Hair Cycle Control

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), and their receptors, GDNF family receptor α-1 (GFRα-1) and GDNF family receptor α-2 (GFRα-2), are critically important for kidney and nervous system development. However, their role in skin biology, specifically in hair growth control, is as yet unknown. We have studied expression and function of GDNF, neurturin, GFRα-1, and GFRα-2 in murine skin during the cyclic transformation of the hair follicle (HF) from its resting state (telogen) to active growth (anagen) and then through regression (catagen) back to telogen. GDNF protein and GFRα-1 messenger RNA are prominently expressed in telogen skin, which lacks NTN and GFRα-2 transcripts. Early anagen development is accompanied by a significant decline in the skin content of GDNF protein and GFRα-1 transcripts. During the anagen-catagen transition, GDNF, GFRα-1, NTN, and GFRα-2 transcripts reach maximal levels. Compared with wild-type controls, GFRα-1 (+/−) and GFRα-2 (−/−) knockout mice show a significantly accelerated catagen development. Furthermore, GDNF or NTN administration significantly retards HF regression in organ-cultured mouse skin. This suggests important, previously unrecognized roles for GDNF/GFRα-1 and NTN/GFRα-2 signaling in skin biology, specifically in the control of apoptosis-driven HF involution, and raises the possibility that GFRα-1/GFRα-2 agonists/antagonists might become exploitable for the treatment of hair growth disorders that are related to abnormalities in catagen development

Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and pertubation of hair follicle cycling

ABSTRACT Lysosomal cysteine proteinases of the papain family are involved in lysosomal bulk proteolysis, major histocompatibility complex class II mediated antigen presentation, prohormone processing, and extracellular matrix remodeling. Cathepsin L (CTSL) is a ubiquitously expressed major representative of the papain‐like family of cysteine proteinases. To investigate CTSL in vivo functions, the gene was inactivated by gene targeting in embryonic stem cells. CTSL‐deficient mice develop periodic hair loss and epidermal hyperplasia, acanthosis, and hyperkeratosis. The hair loss is due to alterations of hair follicle morphogenesis and cycling, dilatation of hair follicle canals, and disturbed club hair formation. Hyperproliferation of hair follicle epithelial cells and basal epidermal keratinocytes–both of ectodermal origin–are the primary characteristics underlying the mutant phenotype. Pathological inflammatory responses have been excluded as a putative cause of the skin and hair disorder. The phenotype of CTSL‐deficient mice is reminiscent of the spontaneous mouse mutant furless(fs). Analyses of the ctsl gene of fs mice revealed a G149R mutation inactivating the proteinase activity. CTSL is the first lysosomal proteinase shown to be essential for epidermal homeostasis and regular hair follicle morphogenesis and cycling.—Roth, W., Deussing, J., Botchkarev, V. A., Pauly‐Evers, M., Saftig, P., Hafner, A., Schmidt, P., Schmahl, W., Scherer, J., Anton‐Lamprecht, I., von Figura, K., Paus, R., Peters, C. Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and pertubation of hair follicle cycling. FASEB J. 14, 2075–2086 (2000

Cathepsin H function in production of lung surfactant proteins.

<p>(<b>A</b>) mRNA expression of surfactant proteins A1, B, and C measured by quantitative ‘real-time’ RT-PCR in lungs of <i>Ctsh^+/+</i> and <i>Ctsh</i>^−/− mice (n = 5 per group). (<b>B</b>) Detection of surfactant protein B (SP-B) in lung tissue lysates (<b>C</b>) Western blot detection of SP-B in broncho-alveolar lavage (BAL) of <i>Ctsh^+/+</i> and <i>Ctsh</i>^−/− mice of 2 genetic backgrounds (129P2/OlaHsd and C57BL/6N). The lysosomal membrane associated protein 2a (Lamp 2a) is present at the limiting membrane of lamellar bodies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026247#pone.0026247-Wasano1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026247#pone.0026247-Albrecht1" target="_blank">[28]</a> and serves as loading control independent of the surfactant proteins. (<b>D</b>–<b>F</b>) Surface activity of BAL fluid measured by pulsating bubble surfactometry (n = 6–10).</p