Forced expression of keratin 16 alters the adhesion, differentiation, and migration of mouse skin keratinocytes

Injury to the skin results in an induction of keratins K6, K16, and K17 concomitant with activation of keratinocytes for reepithelialization. Forced expression of human K16 in skin epithelia of transgenic mice causes a phenotype that mimics several aspects of keratinocyte activation. Two types of transgenic keratinocytes, with forced expression of either human K16 or a K16-C14 chimeric cDNA, were analyzed in primary culture to assess the impact of K16 expression at a cellular level. High K16-C14-expressing and low K16-expressing transgenic keratinocytes behave similar to wild type in all aspects tested. In contrast, high K16-expressing transgenic keratinocytes show alterations in plating efficiency and calcium-induced differentiation, but proliferate normally. Migration of keratinocytes is reduced in K16 transgenic skin explants compared with controls. Finally, a subset of high K16-expressing transgenic keratinocytes develops major changes in the organization of keratin filaments in a time- and calcium concentration-dependent manner. These changes coincide with alterations in keratin content while the steady-state levels of K16 protein remain stable. We conclude that forced expression of K16 in progenitor skin keratinocytes directly impacts properties such as adhesion, differentiation, and migration, and that these effects depend upon determinants contained within its carboxy terminus.     

Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses.

Previously we demonstrated that transgenic mice expressing mutant basal epidermal keratin genes exhibited a phenotype resembling a group of autosomal dominant human skin disorders known as epidermolysis bullosa simplex (EBS). EBS diseases affect approximately 1: 50,000 and are of unknown etiology, although all subtypes exhibit blistering arising from basal cell cytolysis. We now demonstrate that two patients with spontaneous cases of Dowling-Meara EBS have point mutations in a critical region in one (K14) of two basal keratin genes. To demonstrate function, we engineered one of these point mutations in a cloned human K14 cDNA, and showed that a K14 with an Arg-125----Cys mutation disrupted keratin network formation in transfected keratinocytes and perturbed filament assembly in vitro. Since we had previously shown that keratin network perturbation is an essential component of EBS diseases, these data suggest that the basis for the phenotype in this patient resides in this point mutation.     

Overexpression of CRABPI in suprabasal keratinocytes enhances the proliferation of epidermal basal keratinocytes in mouse skin topically treated with all-trans retinoic acid

We investigated whether ectopic expression of CRABPI, a cellular retinoic acid binding protein, influenced the actions of all-trans retinoic acid (ATRA) in transgenic (TG) mice. We targeted CRABPI to the basal vs. suprabasal layers of mouse epidermis by using the keratin 14 (K14) and keratin 10 (K10) promoters, respectively. Greater CRABPI protein levels were detected in the epidermis of adult transgenic(+) mice than in transgenic(-) mice for both transgenes. In adult mouse skin CRABPI overexpression in the basal or suprabasal keratinocytes did not cause morphological abnormalities, but did result in decreased CRABPII mRNA levels. Ectopically overexpressed CRABPI in suprabasal keratinocytes, but not in basal keratinocytes, enhanced the thickening of the epidermis induced by topical ATRA treatments (10 microM, 400 microl for 4 days) by 1.59+/-0.2-fold (p<0.05). ATRA treatment (10 microM) resulted in a 59.9+/-9.8% increase (p<0.05) in the BrdU labeling index in K10/FLAG-CRABPI TG(+) mice vs. TG(-) mice. Retinoid topical treatments reduced p27 and CYP26A1 mRNA levels in TG(+) and TG(-) mouse skin in K14 and K10/FLAG-CRABPI transgenic mice. As epidermal basal keratinocyte proliferation is stimulated by paracrine growth factors secreted by ATRA activated suprabasal keratinocytes, our results indicate that CRABPI overexpression in suprabasal keratinocytes enhances the physiological functions of ATRA.     

Increased Levels of Keratin 16 Alter Epithelialization Potential of Mouse Skin Keratinocytes In Vivo and Ex Vivo

The process of wound repair in adult skin is complex, involving dermal contraction and epithelial migration to repair the lesion and restore the skin's barrier properties. At the wound edge, keratinocytes undergo many changes that engender an epithelialization behavior. The type II keratin 6 and type I keratins 16 and 17 are induced well before cell migration begins, but the role of these proteins is not understood. Forced expression of human K16 in skin epithelia of transgenic mice has been shown to cause dose-dependent skin lesions concomitant with alterations in keratin filament organization and in cell adhesion. Here we show, with the use of a quantitative assay, that these transgenic mice show a delay in the closure of full-thickness skin wounds in situ compared with wild-type and low-expressing K16 transgenic mice. We adapted and validated an ex vivo skin explant culture system to better assess epithelialization in a wound-like environment. Transgenic K16 explants exhibit a significant reduction of keratinocyte outgrowth in this setting. This delay is transgene dose-dependent, and is more severe when K16 is expressed in mitotic compared with post-mitotic keratinocytes. Various lines of evidence suggest that the mechanism(s) involved is complex and not strictly cell autonomous. These findings have important implications for the function of K16 in vivo.     

Directed Expression of a Chimeric Type II Keratin Partially Rescues Keratin 5-null Mice

The crucial role of structural support fulfilled by keratin intermediate filaments (IFs) in surface epithelia likely requires that they be organized into cross-linked networks. For IFs comprised of keratins 5 and 14 (K5 and K14), which occur in basal keratinocytes of the epidermis, formation of cross-linked bundles is, in part, self-driven through cis-acting determinants. Here, we targeted the expression of a bundling-competent KRT5/KRT8 chimeric cDNA (KRT8bc) or bundling-deficient wild type KRT8 as a control to the epidermal basal layer of Krt5-null mice to assess the functional importance of keratin IF self-organization in vivo. Such targeted expression of K8bc rescued Krt5-null mice with a 47% frequency, whereas K8 completely failed to do so. This outcome correlated with lower than expected levels of K8bc and especially K8 mRNA and protein in the epidermis of E18.5 replacement embryos. Ex vivo culture of embryonic skin keratinocytes confirmed the ability of K8bc to form IFs in the absence of K5. Additionally, electron microscopy analysis of E18.5 embryonic skin revealed that the striking defects observed in keratin IF bundling, cytoarchitecture, and mitochondria are partially restored by K8bc expression. As young adults, viable KRT8bc replacement mice develop alopecia and chronic skin lesions, indicating that the skin epithelia are not completely normal. These findings are consistent with a contribution of self-mediated organization of keratin IFs to structural support and cytoarchitecture in basal layer keratinocytes of the epidermis and underscore the importance of context-dependent regulation for keratin genes and proteins in vivo.     

Complementary Roles of Specific Cysteines in Keratin 14 toward the Assembly,Organization, and Dynamics of Intermediate Filaments in Skin Keratinocytes

We recently showed that inter-keratin disulfide bonding plays an important role in the assembly, organization, and dynamics of keratin intermediate filaments in skin keratinocytes. In particular, cysteine 367 located in the central α-helical rod domain of keratin 14 is necessary for the formation of a stable perinuclear network of keratin filaments (with type II partner keratin 5) in skin keratinocytes analyzed by static and live cell imaging. Here, we show that two additional cysteine residues located in the non-helical head domain of K14, Cys-4 and Cys-40, also participate in inter-keratin disulfide bonding and tandemly play a key role complementary to that of Cys-367 in the assembly, organization, and dynamics of keratin filaments in skin keratinocytes in primary culture. Analysis of K14 variants with single or multiple substitutions of cysteine residues points to a spatial and temporal hierarchy in how Cys-4/Cys-40 and Cys-367 regulate keratin assembly in vitro and filament dynamics in live keratinocytes in culture. Our findings substantiate the importance and complexity of a novel determinant, namely inter-keratin disulfide bonding, for the regulation of several aspects of keratin filaments in surface epithelia.     

Transforming growth factor-beta-activated kinase 1 is essential for differentiation and the prevention of apoptosis in epidermis

Transforming growth factor-beta-activated kinase 1 (TAK1) is a member of the mitogen-activated protein (MAP) kinase family and is an upstream signaling molecule of nuclear factor-kappaB (NF-kappaB). Given that NF-kappaB regulates keratinocyte differentiation and apoptosis, TAK1 may be essential for epidermal functions. To test this, we generated keratinocyte-specific TAK1-deficient mice from Map3k7(flox/flox) mice and K5-Cre mice. The keratinocyte-specific TAK1-deficient mice were macroscopically indistinguishable from their littermates until postnatal day 2 or 3, when the skin started to roughen and wrinkle. This phenotype progressed, and the mice died by postnatal day 7. Histological analysis showed thickening of the epidermis with foci of keratinocyte apoptosis and intra-epidermal micro-abscesses. Immunohistochemical analysis showed that the suprabasal keratinocytes of the TAK1-deficient epidermis expressed keratin 5 and keratin 14, which are normally confined to the basal layer. The expression of keratin 1, keratin 10, and loricrin, which are markers for the suprabasal and late phase differentiation of the epidermis, was absent from the TAK1-deficient epidermis. Furthermore, the TAK1-deficient epidermis expressed keratin 16 and had an increased number of Ki67-positive cells. These data indicate that TAK1 deficiency in keratinocytes results in abnormal differentiation, increased proliferation, and apoptosis in the epidermis. However, the keratinocytes from the TAK1-deficient epidermis induced keratin 1 in suspension culture, indicating that the TAK1-deficient keratinocytes retain the ability to differentiate. Moreover, the removal of TAK1 from cultured keratinocytes of Map3k7(flox/flox) mice resulted in apoptosis, indicating that TAK1 is essential for preventing apoptosis. In conclusion, TAK1 is essential in the regulation of keratinocyte growth, differentiation, and apoptosis.     

The human cysteine protease cathepsin V can compensate for murine cathepsin L in mouse epidermis and hair follicles

Mice lacking the ubiquitously expressed lysosomal cysteine protease cathepsin L, show a complex skin phenotype consisting of periodic hair loss and epidermal hyperplasia with hyperproliferation of basal epidermal keratinocytes, acanthosis and hyperkeratosis. The recently identified human cathepsin L-like enzyme cathepsin V, which is also termed cathepsin L2, is specifically expressed in cornea, testis, thymus, and epidermis. To date, in mice no cathepsin V orthologue with this typical expression pattern has been identified. Since cathepsin V has about 75% protein sequence identity to murine cathepsin L, we hypothesized that transgenic, keratinocyte-specific expression of cathepsin V in cathepsin L knockout mice might rescue the skin and hair phenotype. Thus, we generated a transgenic mouse line expressing cathepsin V under the control of the human keratin 14 promoter, which mimics the genuine cathepsin V expression pattern in human skin, by directing it to basal epidermal keratinocytes and the outer root sheath of hair follicles. Subsequently, transgenic mice were crossed with congenic cathepsin L knockout animals. The resulting mice show normalization of epidermal proliferation and normal epidermal thickness as well as rescue of the hair phenotype. These findings provide evidence for keratinocyte-specific pivotal functions of cathepsin L-like proteolytic activities in maintenance of epidermis and hair follicles and suggest, that cathepsin V may perform similar functions in human skin.     

Antagonistic Effects of TGFβ1 and BMP-6 on Skin Keratinocyte Differentiation

Several members of the transforming growth factor β (TGFβ) superfamily are expressed in the developing murine epidermis. Among these are TGFβ1, which is found in the basal layer, and bone morphogenetic protein (BMP)-6, located in the suprabasal layers. Although the role of TGFβ in cell growth has been studied extensively, little is known about the effects of these factors on keratinocyte differentiation. This study demonstrates that BMP-6 acts to positively regulate the differentiation of primary skin keratinocytes grown in culture. In contrast, TGFβ1 antagonizes keratinocyte differentiation blocking the upregulation of keratin markers by BMP-6. We show that the effects of BMP-6 on expression of keratin 1 (K1), a marker of differentiation, requires signaling through the Smad pathway. In addition, regulation of K1 levels by BMP-6 is modulated by the SEK signaling pathway. This suggests that regulation of keratinocyte differentiation by BMP-6 involves multiple signaling systems.     

Human keratin 14 driven HPV 16 E6/E7 transgenic mice exhibit hyperkeratinosis

Human papillomavirus type 16 (HPV16) has been known as a major causative factor for the development of uterine cervical carcinomas. To investigate the in vivo activity of HPV16 expressed in squamous epithelia, transgenic mice harboring HPV16 E6/E7 with human keratin 14 (hK14) promoter were generated. Grossly, hK14 driven HPV16 E6/E7 transgenic mice exhibited multiple phenotypes, including wrinkled skin that was apparent prior to the appearance of hair in neonates, thickened ears, and loss of hair in adults. Transgenic mice with phenotype exhibiting severe wrinkled skin and a lack of hair growth died at the age of 3-4 weeks. Histological analysis revealed that in transgenic mice survived beyond the initial 3-4 weeks, HPV16 E6/E7 causes epidermal hyperplasia in multiple transgenic lineages with high incidence of transgene penetration. This epithelial hyperplasia was characterized by an expansion of the proliferating compartment and keratinocytes, and was associated with hyperkeratosis. Such activities were significantly higher in the skin of transgenic mice than that of the normal mice. Thus, these transgenic mice appeared to be useful for the expression of HPV16 E6/E7 gene and subsequent analysis on hyperkeratosis.     

Expression of a Single,Viral Oncoprotein in Skin Epithelium Is Sufficient to Recruit Lymphocytes

Established cancers are frequently associated with a lymphocytic infiltrate that fails to clear the tumour mass. In contrast, the importance of recruited lymphocytes during premalignancy is less well understood. In a mouse model of premalignant skin epithelium, transgenic mice that express the human papillomavirus type 16 (HPV16) E7 oncoprotein under a keratin 14 promoter (K14E7 mice) display epidermal hyperplasia and have a predominant infiltrate of lymphocytes consisting of both CD4 and CD8 T cells. Activated, but not naïve T cells, were shown to preferentially traffic to hyperplastic skin with an increased frequency of proliferative CD8+ T cells and CD4+ T cells expressing CCR6 within the tissue. Disruption of the interaction between E7 protein and retinoblastoma tumour suppressor protein (pRb) led to reduced epithelial hyperplasia and T cell infiltrate. Finally, while K14E7 donor skin grafts are readily accepted onto syngeneic, non-transgenic recipients, these same skin grafts lacking skin-resident lymphocytes were rejected. Our data suggests that expression of a single oncoprotein in the epidermis is sufficient for lymphocyte trafficking (including immunosuppressive lymphocytes) to premalignant skin.     

Onset of re-epithelialization after skin injury correlates with a reorganization of keratin filaments in wound edge keratinocytes: defining a potential role for keratin 16

Injury to stratified epithelia causes a strong induction of keratins 6 (K6) and 16 (K16) in post-mitotic keratinocytes located at the wound edge. We show that induction of K6 and K16 occurs within 6 h after injury to human epidermis. Their subsequent accumulation in keratinocytes correlates with the profound reorganization of keratin filaments from a pan-cytoplasmic distribution to one in which filaments are aggregated in a juxtanuclear location, opposite to the direction of cell migration. This filament reorganization coincides with additional cytoarchitectural changes and the onset of re-epithelialization after 18 h post-injury. By following the assembly of K6 and K16 in vitro and in cultured cells, we find that relative to K5 and K14, a well- characterized keratin pair that is constitutively expressed in epidermis, K6 and K16 polymerize into short 10-nm filaments that accumulate near the nucleus, a property arising from K16. Forced expression of human K16 in skin keratinocytes of transgenic mice causes a retraction of keratin filaments from the cell periphery, often in a polarized fashion. These results imply that K16 may not have a primary structural function akin to epidermal keratins. Rather, they suggest that in the context of epidermal wound healing, the function of K16 could be to promote a reorganization of the cytoplasmic array of keratin filaments, an event that precedes the onset of keratinocyte migration into the wound site.     

Srcasm corrects Fyn-induced epidermal hyperplasia by kinase down-regulation

Src family tyrosine kinases (SFKs) are important regulators of epithelial cell growth and differentiation. Characterization of cellular mechanisms that regulate SFK activity will provide insights into the pathogenesis of diseases associated with increased SFK activity. Keratin 14-Fyn (K14) transgenic mice were derived to characterize the effect of Fyn on epidermal growth and differentiation in vivo. The epidermis of K14-Fyn mice is thickened, manifests prominent scale, and exhibits features consistent with hyperproliferation. Increased epidermal Fyn levels correlate with activation of p44/42 MAP kinases, STAT-3, and PDK-1, key signaling molecules that promote epithelial cell growth. The Src-activating and signaling molecule (Srcasm) is a substrate of SFKs that becomes tyrosine-phosphorylated downstream of the EGF receptor. In vitro, increased Srcasm levels promote activation of endogenous Fyn and keratinocyte differentiation. To study the in vivo effect of Srcasm upon Fyn, double transgenic lines were derived. K14-Fyn/Srcasm transgenic mice did not manifest the hyperproliferative phenotype. In contrast, K14-Fyn/Srcasm-P transgenic mice, which express a nonphosphorylatable Srcasm mutant, maintained the hyperproliferative phenotype. Resolution of the hyperproliferative phenotype correlated with reduced Fyn levels in vivo in three experimental systems: transgenic mice, primary keratinocytes, and cell lines. Biochemical studies revealed that Srcasm-dependent Fyn down-regulation requires Fyn kinase activity, phosphorylation of Srcasm, and the Srcasm GAT domain. Therefore, Srcasm is a novel regulator of Fyn promoting kinase down-regulation in a phosphorylation-dependent manner. Srcasm may act as a molecular "rheostat" for activated SFKs, and cellular levels of Srcasm may be important for regulating epithelial hyperproliferation associated with increased SFK activity.     

Progressive squamous epithelial neoplasia in K14-human papillomavirus type 16 transgenic mice.

To model human papillomavirus-induced neoplastic progression, expression of the early region of human papillomavirus type 16 (HPV16) was targeted to the basal cells of the squamous epithelium in transgenic mice, using a human keratin 14 (K14) enhancer/promoter. Twenty-one transgenic founder mice were produced, and eight lines carrying either wild-type or mutant HPV16 early regions that did not express the E1 or E2 genes were established. As is characteristic of human cancers, the E6 and E7 genes remained intact in these mutants. The absence of E1 or E2 function did not influence the severity of the phenotype that eventually developed in the transgenic mice. Hyperplasia, papillomatosis, and dysplasia appeared at multiple epidermal and squamous mucosal sites, including ear and truncal skin, face, snout and eyelids, and anus. The ears were the most consistently affected site, with pathology being present in all lines with 100% penetrance. This phenotype also progressed through discernible stages. An initial mild hyperplasia was followed by hyperplasia, which further progressed to dysplasia and papillomatosis. During histopathological progression, there was an incremental increase in cellular DNA synthesis, determined by 5-bromo-2'-deoxyuridine incorporation, and a profound perturbation in keratinocyte terminal differentiation, as revealed by immunohistochemistry to K5, K14, and K10 and filaggrin. These K14-HPV16 transgenic mice present an opportunity to study the role of the HPV16 oncogenes in the neoplastic progression of squamous epithelium and provide a model with which to identify genetic and epigenetic factors necessary for carcinogenesis.     

Discovery of keratin function and role in genetic diseases: the year that 1991 was

In 1991, a set of transgenic mouse studies took the fields of cell biology and dermatology by storm in providing the first credible evidence that keratin intermediate filaments play a unique and essential role in the structural and mechanical support in keratinocytes of the epidermis. Moreover, these studies intimated that mutations altering the primary structure and function of keratin filaments underlie genetic diseases typified by cellular fragility. This Retrospective on how these studies came to be is offered as a means to highlight the 25th anniversary of these discoveries.Although intermediate filaments (IFs) have been characterized at some level for a longer period of time (Oshima, 2007 ), they were officially discovered as such as recently as 1968 by Howard Holtzer and colleagues while studying the developing skeletal muscle (Ishikawa et al., 1968 ). The advent of gene cloning methods and monospecific antibody production in the late 1970s and throughout the 1980s led to an explosion of data and knowledge about IFs that established them as a large family of genes and proteins that are individually regulated in a tight and evolutionarily conserved tissue- and differentiation-specific manner. Researchers also uncovered some of the remarkable properties of IFs as purified elements in vitro and in living systems and recognized that they occur in the nucleus as well as in the cytoplasm. In spite of the fast pace of progress during that period, however, it was not possible to produce evidence that spoke unequivocally about the functional importance of IFs in cells and tissues, let alone their role in disease.Beginning in the mid- to late 1980s, pioneering experimentation along two distinct lines was underway in the laboratory of Elaine Fuchs, then at the University of Chicago. The eventual merger of these approaches yielded the first formal insight into IF function in vivo, as well as into their direct involvement in human disease. In an effort to define structure–function relationships with regard to the assembly and network formation properties of IFs, one such approach was the application of systematic deletion mutagenesis to keratin 14 (K14), a type I IF that is expressed with its type II partner keratin 5 (K5) in the progenitor basal layer of the epidermis and related complex epithelia. These studies demonstrated that deleting sequences from either end of the central α-helical rod domain of the K14 protein was deleterious for filament formation in a dominant manner both in transfected cells (Albers and Fuchs, 1987 , 1989 ; Figure 1) and the setting of IF polymerization assays involving purified proteins in vitro (e.g., Coulombe et al., 1990 ). The second key effort in the Fuchs lab in the late 1980s resulted in the demonstration that the proximal 2.5 kb and distal 700 base pairs corresponding respectively to the 5′ upstream and 3′ downstream regions of the cloned human K14 gene were sufficient to confer tissue-specific, that is, K14-like, regulation in transgenic mice in vivo (Vassar et al., 1989 ; Figure 1). This tour de force paved the way for the production of a human K14 gene promoter–based cassette (e.g., Saitou et al., 1995 ) that could reliably direct the expression of any open reading frame in a K14-like manner in transgenic mice. As an aside, this tool has had a profound effect on epithelial and skin biology research.Open in a separate windowFIGURE 1:Schematic representation of the strategy and outcome of the experiments that led to the discovery of keratin function and role in genetic disease. Original figures are reproduced to give a realistic account of the data. (A) Examples of a disrupted keratin filament network in cultured epithelial cells transfected with and expressing a dominantly acting K14 deletion mutant (arrows). (Reproduced from Albers and Fuchs, 1987 , with permission.) (B) Preferential expression of a substance P-epitope–tagged transgenic human K14 protein in the basal layer of tail skin epidermis in mouse, conveying the tissue- and differentiation-specific behavior of the transgene. (Reproduced from Vassar et al., 1989 , with permission.) (C) The two experimental approaches described in A and B were combined to assess the consequences of tissue-specific expression of dominantly acting K14 mutants in skin tissue in vivo. (D) Newborn mouse littermates. The mouse at the top is transgenic (Tg) and expresses a mutated form of K14 in the epidermis. It is showing severe skin blistering (arrows), particularly in its front paws, which are heavily used by mouse newborns to feed from their mother. The bottom mouse is a nontransgenic control showing no such blistering. (E, F) Hematoxylin-eosin–stained skin tissue sections showing the location of subepidermal cleavage within the epidermis of a K14 mutant–expressing transgenic mouse (opposing arrows in E). Cleavage occurs at the level of the basal layer, where the mutant keratin is expressed. Again, this is never seen in control wild-type (Wt) skin (F). Bar, 100 μm (E, F). (D–F are from Coulombe et al., 1991b , with permission.) (G) Leg skin in a patient suffering from the Dowling–Meara form of epidermolysis bullosa simplex. Characteristic of this severe variant of this disease, several skin blisters are often grouped in a herpetiform manner (Fine et al., 1991 ).Subsequent use of the human K14 promoter–based cassette to direct the expression of epitope-tagged and selected deletion mutants of K14 gave rise to transgenic mouse pups that exhibited extensive blistering of the skin preferentially at sites of frictional trauma (Coulombe et al., 1991b ; Vassar et al., 1991 ; Figure 1). Electron microscopy showed that skin blistering occurred secondary to a loss of the integrity of keratinocytes located in the basal layer of the epidermis, that is, the precise site of mutant K14 protein accumulation. Such blistering did not occur in transgenic mice expressing a full-length version of human K14 modified to carry only an epitope tag at the C-terminus at similar or higher levels (Coulombe et al., 1991b ; Vassar et al., 1991 ). In addition, the severity of skin blistering in mutant K14–expressing transgenic pups could be directly related to the extent to which the mutant protein had been shown to disrupt filament assembly in transfected cell assays and in IF reconstitution assays in vitro. For instance, tissue-specific expression of a K14 mutant that could severely disrupt 10-nm filament assembly was associated with whole-body skin blistering and the untimely death of mouse pups and, from a pathology perspective, with “tonofilament clumping” and a paucity of visible keratin IFs in transgenic basal keratinocytes. By comparison, expression of another K14 mutant with a less deleterious effect on 10-nm IF assembly was compatible with the survival of transgenic mouse pups and resulted in skin blistering largely limited to the front paws in newborn mice together with altered organization of keratin IFs in basal keratinocytes of transgenic epidermis in situ, albeit without tonofilament clumping. This initial set of mouse strains thus revealed the existence of a direct link between the so-called “genotype” (i.e., mutant K14 characteristics) and the skin phenotype (Coulombe et al., 1991b ; Vassar et al., 1991 ; Fuchs and Coulombe, 1992 ). Electrophoretic analyses of protein samples confirmed that the K14 mutant proteins acted dominantly to produce such spectacular phenotypes in transgenic mouse skin. Finally, blistering also occurred in the mutant K14–expressing transgenic mice in other stratified epithelia known both to express K14 and experience trauma, notably in the oral mucosa (Coulombe et al., 1991b ; Vassar et al., 1991 ).It is worth celebrating the 25th anniversary of these pioneering experiments for the following two reasons. First, the study of these mice provided the first formal demonstration that keratin IFs play a fundamentally important role in structural support in surface epithelia such as the epidermis and oral mucosa. Without proper IF support, epidermal keratinocytes are rendered fragile and cannot sustain trivial frictional stress (Coulombe et al., 1991b ; Fuchs and Coulombe, 1992 ). The second reason is the observation that the phenotype of these K14 mutant–expressing mice proved eerily similar to those of individuals afflicted with the disease epidermolysis bullosa simplex (EBS), a rare, dominantly inherited and debilitating skin condition in which the epidermis and oral mucosa undergo blistering after exposure to trivial mechanical trauma. As observed in the mouse model, tissue cleavage had been shown to result from the loss of integrity of keratinocytes located in the basal layer (Fine et al., 1991 ). Further, other researchers had previously reported on anomalies in the organization of keratin IFs in the basal epidermal keratinocytes of EBS patients (Anton-Lamprecht, 1983 ; Ito et al., 1991 ) or in cultures of epidermal keratinocytes established from EBS patients (Kitajima et al., 1989 ). The Fuchs laboratory thus teamed up with Amy Paller, a physician-scientist and pediatric dermatologist with deep expertise in genodermatoses, and mutations were soon discovered in the K14 gene of two independent and sporadic cases of a severe variant of the disease known as Dowling–Meara EBS (Coulombe et al., 1991a ; Figure 1). The two mutations were heterozygous missense alleles that affected the very same codon in K14 (Arg-125) and were correctly predicted at the time to correspond to a mutational hot spot in type I keratin genes. The mutations were shown to dominantly disrupt 10-nm IF assembly in vitro and/or in transfected keratinocytes in culture (Coulombe et al., 1991a ). Soon thereafter, a team led by Ervin Epstein at University of California, San Francisco (San Francisco, CA), reported on the use of classical linkage analysis to uncover a missense mutation in the K14 gene of a small pedigree with Koebner-type EBS, a less severe variant of the disease (Bonifas et al., 1991 ). The next year, Birgit Lane and colleagues (Lane et al., 1992 ) reported on the occurrence of mutations in keratin 5 (K5), the formal type II keratin assembly partner for K14 in vivo, in another instance of Dowling–Meara EBS.In the years since 1991, a role in structural support has been formally demonstrated for all classes of IFs (Coulombe et al., 2009 ), including the nuclear-localized lamins (e.g., Lammerding et al., 2004 ). Moreover, we now know of several hundred independent instances of mutations in either K5 or K14 in the setting of the EBS disease, with the vast majority of those consisting of dominantly acting missense alleles (Szeverenyi et al., 2008 ; Human Intermediate Filament Database, www.interfil.org, maintained at the Centre for Molecular Medicine and Bioinformatics Institute, Singapore). We also learned that, as anticipated, EBS largely represents a loss-of-function phenotype, since K14-null mice (Lloyd et al., 1995 ), K14-null individuals (Chan et al., 1994 ; Rugg et al., 1994 ), and K5-null mice (Peters et al., 2001 ) all exhibit an EBS-like skin-blistering phenotype (Coulombe et al., 2009 ). Mutations such as Arg125Cys in K14 markedly compromise the remarkable mechanical properties of keratin filaments (Ma et al., 2001 ), as well as the steady-state dynamics of keratin filaments in transfected keratinocytes in culture (Werner et al., 2004 ). Finally, mutations affecting the coding sequence of IF genes have been shown to underlie >100 diseases affecting the human population (Omary et al., 2004 ; Szeverenyi et al., 2008 ; www.interfil.org). Consistent with the exquisite tissue- and cell type–specific regulation of IF genes, these diseases collectively affect a myriad of tissues and organs and are relevant to nearly all branches of medicine. These observations attest to the importance and profound effect that the generation and characterization of mutant K14–expressing transgenic mice has had for cell biology, epithelial physiology, dermatology, and medicine.Many thoughts spring to mind when reminiscing about my involvement with this body of work. First, this effort was prescient of the power of team science and, in particular, of the potential effect of close collaborations involving biologists and physician-scientists. I learned a great deal and benefited immensely from working closely with many colleagues on this project, including Bob Vassar, Kathryn Albers, Linda Degenstein, Liz Hutton, Anthony Letai, Amy Paller, and, last but not least, my postdoctoral mentor and the laboratory head, Elaine Fuchs. Second, there is no substitute for elements such as innovation, hard work, perseverance, boldness, accountability, and great leadership. Elaine had the vision and created the exceptional circumstances necessary to make this set of discoveries possible, and, of equal importance, she was an integral part of the day-to-day progress and maturation of the entire project. Finally, as we all know, there is an intangible element of luck involved in discovery research. In this instance, a strong argument can be made that the studies highlighted here may not have had such a deep and defining effect had the effort been devoted to any IF other than the K5–K14 keratin pairing.What are some of the lingering issues regarding this specific topic that preoccupy us still, 25 years later? Two challenges loom particularly large. First, we have yet to achieve a satisfactory understanding of how mutations in keratin proteins can cause disease. This is due in part to the lack of an atomic-level understanding of the core structure of IFs (which has been a tough nut to crack; Lee et al., 2012 ), along with the reality that, for any relevant IF gene, there is a broad variety of disease-associated (mostly missense) mutations that pepper their primary structure (www.interfil.org). Second, we have yet to achieve success toward the treatment of EBS or any IF-based disorder. Disease characteristics such as low incidence, a dominantly inherited character, genetic heterogeneity (e.g., broad mutational landscape), and, in the case of EBS and related conditions, an intrinsically high rate of cell turnover within the main target tissue significantly add to the challenge of devising safe and effective therapeutic strategies (Coulombe et al., 2009 ). Although efforts are still underway to foster progress on these two challenging issues, the field as a whole has made significant progress in uncovering a plethora of noncanonical functions of keratin IFs (Hobbs et al., 2016 ) in addition to understanding their regulation, dynamics, and many remarkable properties.     

Formation of a Normal Epidermis Supported by Increased Stability of Keratins 5 and 14 in Keratin 10 Null Mice

The expression of distinct keratin pairs during epidermal differentiation is assumed to fulfill specific and essential cytoskeletal functions. This is supported by a great variety of genodermatoses exhibiting tissue fragility because of keratin mutations. Here, we show that the loss of K10, the most prominent epidermal protein, allowed the formation of a normal epidermis in neonatal mice without signs of fragility or wound-healing response. However, there were profound changes in the composition of suprabasal keratin filaments. K5/14 persisted suprabasally at elevated protein levels, whereas their mRNAs remained restricted to the basal keratinocytes. This indicated a novel mechanism regulating keratin turnover. Moreover, the amount of K1 was reduced. In the absence of its natural partner we observed the formation of a minor amount of novel K1/14/15 filaments as revealed by immunogold electron microscopy. We suggest that these changes maintained epidermal integrity. Furthermore, suprabasal keratinocytes contained larger keratohyalin granules similar to our previous K10T mice. A comparison of profilaggrin processing in K10T and K10(-/-) mice revealed an accumulation of filaggrin precursors in the former but not in the latter, suggesting a requirement of intact keratin filaments for the processing. The mild phenotype of K10(-/-) mice suggests that there is a considerable redundancy in the keratin gene family.     

Suppression of keratin 15 expression by transforming growth factor beta in vitro and by cutaneous injury in vivo

Transforming growth factor beta (TGF-beta) is a multifunctional cytokine which plays an important role in cutaneous wound repair. To gain insight into the mechanisms of action of this growth and differentiation factor in the skin, we searched for genes which are regulated by TGF-beta1 in cultured HaCaT keratinocytes. Using the differential display RT-PCR technology we identified a gene which was strongly downregulated by TGF-beta1. The identified cDNA includes sequences of the keratin 15 (K15) gene which encodes a component of the cytoskeleton of basal cells in stratified epithelia. Surprisingly, our cDNA also included an unknown sequence. Since this cDNA lacks an open reading frame, the corresponding mRNA is likely to be nonfunctional. However, we also demonstrate a strong negative regulation of the expression of the published, functional K15 variant. Expression of K15 was also suppressed by tumor necrosis factor alpha (TNF-alpha) and to a lesser extent by epidermal growth factor (EGF) and keratinocyte growth factor (KGF). By contrast, the major basal type I keratin, K14, was upregulated by TGF-beta1, whereas TNF-alpha, EGF, and KGF had no effect. Consistent with the in vitro data, we found a significant reduction of the K15 mRNA levels after skin injury, whereas K14 expression increased during the wound healing process. Immunostaining revealed the presence of K15 in all basal cells of the epidermis adjacent to the wound, but not in the hyperproliferative epithelium above the granulation tissue. These data demonstrate that K15 is excluded from the activated keratinocytes of the hyperthickened wound epidermis, possibly as a result of increased growth factor expression in injured skin.     

Targeted somatic mutagenesis in mouse epidermis

Gene targeting in the mouse is a powerful tool to study mammalian gene function. The possibility to efficiently introduce somatic mutations in a given gene, at a chosen time and/or in a given cell type will further improve such studies, and will facilitate the generation of animal models for human diseases. To create targeted somatic mutations in the epidermis, we established transgenic mice expressing the bacteriophage P1 Cre recombinase or the tamoxifen-dependent Cre-ER(T2) recombinase under the control of the human keratin 14 (K14) promoter. We show that LoxP flanked (floxed) DNA segments were efficiently excised in epidermal keratinocytes of K14-Cre transgenic mice. Furthermore, Tamoxifen administration to adult K14-Cre-ER(T2) mice efficiently induced recombination in the basal keratinocytes, whereas no background recombination was detected in the absence of ligand treatment. These two transgenic lines should be very useful to analyse the functional role of a number of genes expressed in keratinocytes.     

The expression of keratin k10 in the basal layer of the epidermis inhibits cell proliferation and prevents skin tumorigenesis

Forced expression of K10, a keratin normally expressed in postmitotic, terminally differentiating epidermal keratinocytes, inhibits the progression of the cell cycle in cultured cells (Paramio, J. M., Casanova, M. Ll., Segrelles, C., Mittnacht, S., Lane, E. B., and Jorcano, J. L. (1999) Mol. Cell. Biol. 19, 3086-3094). This process requires a functional retinoblastoma (pRb) gene product and is mediated by K10-induced inhibition of Akt and PKCzeta, two signaling intermediates belonging to the phosphoinositide (PI) 3-kinase signal transduction pathway (Paramio, J. M., Segrelles, C., Ruiz, S., and Jorcano, J. L. (2001) Mol. Cell. Biol. 21, 7449-7459). Extending earlier in vitro studies to the in vivo situation, this work analyzes the alterations found in transgenic mice that ectopically express K10 in the proliferative basal cells of the epidermis. Increased expression of K10 led to a hypoplastic and hyperkeratotic epidermis due to a dramatic decrease in skin keratinocyte proliferation in association with the inhibition of Akt and PKCzeta activities. The inhibition of cell proliferation and Akt and PKCzeta activities was also observed although to a minor extent in low hK10-expressing mice. These animals displayed no overt epidermal phenotype nor overexpression of K10. In these non-phenotypic mice, ectopic K10 expression also resulted in decreased skin tumorigenesis. Collectively, these data demonstrate that keratin K10 in vivo functions include the control of epithelial proliferation in skin epidermis.