Keratins in Skin Epidermal Development and Diseases

Epidermal keratinocyte (KC), the major cell type in the skin epidermis, plays critical roles in forming a permeability barrier to separate internal organs from external stimuli. Keratins, constituting about 30–80% of the total protein in KCs, form the major intermediate filament cytoskeleton of KC. Keratins consist of 54 unique genes in humans and they are expressed in cell-, differentiationand development-dependent manner. While keratin pairs K5-K14 and K1-K10 are normally associated with KCs at different cell differentiation stages, other keratin pairs such as K6-K16/K17 and K8–K18 and are usually not expressed in normal skin interfollicular epidermis, but are elevated during wounding, inflammatory skin diseases such as psoriasis or malignant conversion of KC. The expression and function of keratins are tightly regulated at both transcriptional and post-transcriptional levels. Inherited or spontaneous mutations in keratins or abnormal keratin regulations or modifications can cause KC and cutaneous tissue fragility, skin hypertrophic and inflammatory conditions or malignant transformation of KC, therefore accounting for a large number of disorders in human skin. Here we review the recent literature on how keratins are normally expressed during skin development and how mutations or misregulations of these keratins are involved in the pathogenesis of skin diseases.

protein and forming the major intermediate filament cytoskeleton of the epidermis. Since keratin family proteins were initially characterized based on their mobility on 2D SDS-PAGE back in 1980s, more than 50 mammalian keratins have been identified and characterized. Keratins can be sub-classified into two distinct classes: Type I keratins, including K9-K40, are relative acidic (pKi = 4.5-5.5) and small  whereas type II keratins, including K1-K8 and K71-86), are more basic (pKi = 5.5-7.5) and larger (53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(66)(67) [1][2][3]. The active keratin genes are clustered into two dense region of the chromosome: all type II keratins plus one type I keratin (K18) are located on chromosome 12q, and the remaining type I keratins are all on chromosome 17q. Despite the fact that type I and type II keratins are located at distinct region of the chromosome, they show beautifully specific patterns of gene expression within adjacent epidermal cell layers and a specific pair of keratins are usually co-expressed as a heterodimer between one acidic (type I) and one basic (type II) keratin [4]. These keratin heterodimers selfassemble into antiparallel, staggered tetramers, yielding intermediate filament through lateral and longitudinal interactions [4].
The primary function of the keratin intermediate filament cytoskeleton is to provide cells with structural resilience against mechanical trauma, and this is especially important for epidermal cells because as the outermost barrier tissue the epidermis has to have the ability to resist some of the most severe physical stress levels experienced by any human tissue. The basic structural organization of keratin intermediate filaments contains four helical or coiledcoil segments flanked by N-and C-terminal glycine rich sequence. And most disease-causing mutations occur in the well-conserved coiled-coil domains of keratins, leading to disruption of secondary structure formation between the heterodimer and the subsequent aggregation of the keratin filaments. In addition to structural and mechanical support, cell-specific keratin expression also modulates growth, adhesion, migration and invasion of epithelial cells. Thus, dysfunction or mutations of keratin proteins are associated with a remarkable variety of skin disorders, such as skin blistering, inflammatory disorders and skin tumors. So far more than 100 different disorders (termed as keratinopathies) have been linked to inherited keratin changes (www.interfil.org).
In this chapter we will first describe how keratins are normally expressed and regulated during epidermal development and in adult homeostasis, then we will describe how mutation or abnormal expression or modification of keratin proteins are associated with various skin diseases, and their function and regulatory mechanisms during disease pathogenesis. Keratin mutations and related diseases in skin appendages, including hair and nail, will not be reviewed here.

Keratin expression kinetics during epidermal development
The barrier function of epidermis is mainly provided by keratinocytes (KC), the predominant cell type in the epidermis, and it is maintained by a tightly controlled balance between proliferation and differentiation of KC [5,6]. As shown in Figure 1, the murine epidermal development begins at embryonic day (E) 8.5 from a single layer of progenitor cells that express keratin pair K8-K18, and around E 9.5 these progenitor cells are specified to an epidermal cell fate and switch to express a different pair of keratins, K5-K14 [7][8][9][10]. Between E 10.5-E15.5, the committed KC begin a process of upward stratification and differentiation leading to the generation of suprabasal cells, in which cell cycle is arrested and early differentiation program is initiated and K5/K14 are substituted by K1 and K10. Studies have suggested that the ectopic expression of K10 inhibits cell cycle progression and proliferation and thus promotes terminal differentiation of keratinocytes [11,12]. At E 16.5, the suprabasal KC commit to terminal differentiation and continue to migrate upward forming the granular layer, and these granular KCs express late differentiation markers, such as Filaggrin (FLG), Loricrin (LOR) and Involucrin (INV). By E 18.5, epidermis becomes fully mature, and terminally differentiated KCs become enucleated corneocytes forming the outer most cornified layer with complete barrier function.
Note that K15 is an additional type I keratin protein co-expressed with K5/K14 in the basal keratinocytes of stratified epithelia and in the bulge in hair follicles [13], and K2 is an additional type II keratin protein co-expressed with K1/K10 in the differentiated keratinocytes [14]. Although K15 or K2 are generally considered as minor keratins, but the ratio of K15 to K14 or K2 to K1 can vary dramatically during development or upon disease condition at different skin sites [15]. In adult skin, K15 expression is restricted to hair follicle stem cells [16,17]. This cellstage specific expression pattern of keratins is precisely maintained through postnatal development and adulthood under homeostatic condition in both human and mouse skin epidermis. Therefore K5 and/or K14 are highly specific markers for basal proliferative KC, K15 is generally used as a marker for epithelial stem cells in the hair follicle bulge, and K1 and/or K10 have been robustly used to mark KC in the early differentiation stage (Figure 2).

Regulation of keratin expression during development
Keratin synthesis is regulated at the level of transcription by a characteristic constellation of transcription factors. Regulatory mechanisms for the expression of K5 and K14, the key keratins forming the cytoskeletal IF network in mitotically active basal cells, have been extensive studied. Studies have shown that the promoter activities of K5 and K14 genes are collaboratively regulated by several transcription factors, including AP1, AP2, NFκB, Skn-1a, Tst-1, RAR (nuclear receptor for retinoic acid), T3R (receptor for thyroid hormone), GR (glucocorticoid receptor) and coactivator CBP/p300 in response to many extracellular signals, such as growth factor, vitamins (retinoic acid/VitA), thyroid hormone, or glucocorticoids [18][19][20][21][22] .
The expression of K1 and K10 is upregulated during early differentiation process. Forced expression of transcription factor C/EBPβ in keratinocytes arrested growth and induced the expression of K1 and K10 but had a minimal effect on the expression of late differentiation markers [23]. In consistent, Cebpb-deficient mice had reduced levels of K1 and K10 but not of Lor or Inv, suggesting that C/EBPβ modulates K1 and K10 expression during early events of keratinocyte differentiation. Other transcription factors, such as C/EBPα and AP2 are also required for K10 expression during differentiation [24]. Therefore, C/EBPβ, C/EBPα and AP2 are considered the differentiation-associated transcription factors controlling early differentiation process of keratinocytes. We have shown previously that in cultured primary mouse basal keratinocytes, while elevating extracellular calcium robustly triggers stratification and the expression of late differentiation markers, K10 expression is only moderately increased by high calcium [6,25]. In contrast, growth factor depletion/starvation strongly induced K10 expression [6,25], suggesting that growth arrest but not high calcium is the key signal to turn on K10 expression. Indeed, studies have shown that C/EBPβ expression localizes in the upper Keratin differentiated layers of human skin epidermis [26] and C/EBP family of transcription factors inhibit proliferation by blocking cell cycle progression [27,28]. Therefore it is possible that growth factor depletion condition in culture induces and activates C/EBPs which in turn trigger cell cycle arrest and induction of K10 gene.
K8 and K18 are the keratin pair that is expressed earliest during embryonic development of the epidermis and their expressions are suppressed upon epidermal progenitor cells commitment to keratinocyte lineage [8,29]. Several transcription factors, such as p63, Ovol2 and Ctip2, have been shown to suppress K8 expression during epidermal development [6,9,30]. It has been shown that developing murine p63À/À epidermis expressed high level of K8 and failed to develop a fully mature stratified epidermis [9], suggesting that p63 may regulate epidermal development through suppressing K8 transcription. We have also shown that in wild-type mouse keratinocytes K8 promoter was repressed by transcription factor Ctip2, and upon Ctip2 depletion K8 expression became strongly upregulated in both developing epidermis and in primary mouse keratinocytes at both transcript and protein levels, demonstrating that Ctip2 functions as a transcription suppressor of K8 gene [6].

Epidermolysis bullosa simplex diseases and K5/K14
Epidermolysis bullosa simplex (EBS) is an autosomal dominant skin disorder, manifests itself upon trauma in the form of epidermal basal cell death leading to skin blisters [31]. The primary cause of EBS is dominant mutations in either of the genes encoding keratins K5 or K14 [32]. This pair of type I (K14) and type II (K5) is specifically expressed in the basal cell layer of the skin epidermis, which is in direct contact with the basement membrane of the underlying dermis. Therefore, loss of function or mutation of K5 or K14 leads to defects in keratin filament formation in basal keratinocytes and thus triggers skin blistering due to fragility of the basal cell compartment in either human or mouse skin epithelium [33][34][35] (Figure 3). Additionally, it has been shown that overexpression of transcription factor Ovol2 in mouse basal keratinocytes caused reduced K5 and K14 expression, leading to a severe blistering phenotype that resembles the clinical features of EBS [30].
Although K5/K14 mutations are associated with the majority of EBS population, about a quarter of patients with EBS do not have genomic mutations nor abnormal transcript expressions of K5 or K14 [36], suggesting that alternative pathways may be involved in the pathogenesis of these patients. Indeed, recent studies have suggested that in addition to genetic mutation, abnormal K5/K14 functions in basal keratinocytes can also be regulated at posttranscriptional level through post-translational modifications, such as phosphorylation and disulfide bonding. Abnormal phosphorylation of keratins contributes to the pathogenesis and progression of EBS [37]. For example, threonine 150 (T150) in the head domain of K5 was found to be phosphorylated in human EBS keratinocytes. Expression of phosphomimetic T150D K5 mutant in keratinocytes arrested keratin heterodimer assembly leading to impaired keratin filament formations and reduced cell viability and elevated response to stressors [38], suggesting a possible role of K5 T150 phosphorylation in EBS pathogenesis. In addition to phosphorylation, it has been shown that proper disulfide bonding between K14-K5 heterodimers is required to maintain keratin intermediate filament organization and dynamics in primary mouse skin keratinocytes, and disruption of this K14-dependent disulfide linkages may lead to keratinopathies, such as EBS [39].

Epidermolytic hyperkeratosis diseases and K1/K10
When basal keratinocytes migrate to suprabasal layer of the epidermis, supra basal cells cease transcription of K5 and K14 but instead express K1 and K10. Mutations in either K1 or K10 cause several human skin diseases, such as Epidermolytic ichthyosis (EI), Bullous ichthyosis (BI), palmar-plantar keratoderma and Epidermolytic nevus (EN). BI/EI are caused by rare autosomal dominant mutations of either of K1 or K10 that manifest at birth with fragile blisters and erosions that develop into hyperkeratotic lesions. X-ray crystal structure analysis of K1-K10 heterodimer suggested that point mutation of these keratins may disrupt the disulfide linkage and secondary structure formation between the heterodimer leading to aggregation of the keratin filaments [40]. In contrast to EBI, only the K1 or K10 expressing suprabasal cells are affected in EI/BI patients, and the basal proliferative compartment are not affected. However, these unaffected basal proliferating cells are bathed beneath the rupturing suprabasal cells with inflammatory cytokines, leading to over-proliferation of the basal cells and epitheliumknown as hyperkeratosis (Figure 3). Therefore, BI skin contains a highly thicken epidermis made up of fragile cells and it is highly susceptible to bacterial and fungal colonization and is highly disfiguring and debilitating for the patient.
In contrast to EI or BI, Epidermolytic nevus (EN) is caused by somatic mutation of either K1 or K10, and the skin blistering phenotype is only affecting part of the body [41,42]. Because germ line cells are not affected in EN patient, EN parents usually do not transmit the mutations and disease to next generation. However, it has been reported that under rare conditions EN can produce EI in the next generation through transmission from mosaic to germ line. And the risk of disease transmission to the next generation can be evaluated by next generation sequencing of mutation rate in sperm, leukocytes and lesional skin of the EN patients who wish to bear children [43].

Superficial epidermolytic ichthyosis (SEI) and K2
Superficial epidermolytic ichthyosis (SEI), previously known as ichthyosis bullosa of Siemens (IBS), is an autosomal dominant skin disorder linked to K2 mutations and it is characterized by superficial epidermal fragility and desquamation that lead to characteristic denuded areas. In SEI, aggregates of KF bundles and cytolysis are confined to the upper spinous and granular layers of the epidermis where K2 is expressed [44,45]. In human, K2 is expressed later in differentiation in the upper spinous and granular layers of skin collected from different body sites [14], but SEI patients usually develop more severe symptoms in palms and soles compared to other body sites suggesting that K2 may play a major role to support tissue integrity in these areas. The tissue specific expression pattern of K2 has been better characterized in mouse skin: in regions of the soles (except foodpad which expresses K2-K9/K10), ears and tail of the mouse, K2 instead of K1 is the major type II keratin that pairs with K10. K2À/-K10À/À double knockout mice or K2À/Àmice developed epidermal acanthosis and hyperkeratosis in the tail epidermis, ear epidermis and inter-footpad epidermis of the soles [46,47], demonstrating that K2-K10 keratin pairs are essential for the epidermal integrity of plantar skin.

Epidermal palmoplantar keratoderma (EPPK) and K9
Epidermal palmoplantar keratoderma (PPK) is an autosomal dominant skin disorder that develops shortly after birth, and manifests as diffuse hyperkeratosis of the palms and soles and showing sharp demarcations with erythematous margin. Mutations of K9, which is expressed specifically in the suprabasal keratinocytes of the glabrous skin epidermis (palms and soles), are the major cause for EPPK [48,49]. In human plantar and palmar epidermis, K9 functions as the additional type I keratin, besides K10, to partner with type II keratin K1 [50]. Therefore, mutations of K9 cause pathological epidermal thickening on palms and soles, manifesting as different forms of palmoplantar keratodermas [49]. In mice, K9 expression is restricted to skin epidermis of the footpad, and K9 deficient mice developed calluses marked by hyperpigmentation that are exclusively localized to the stress-bearing footpad. Additionally, hyperproliferation, impaired terminal differentiation, and abnormal expression of K5, K14 and K2 were found in the lesions of K9 deficient mice [51]. Together, these evidence demonstrate that K9 is required for the structural integrity and terminal differentiation of the palmoplantar epidermis.

Skin wounds and inflammatory skin diseases and K6/K16/K17
In mammals, the skin of the palm is uniquely adapted to withstand remarkable physical stress, and the palmoplantar epidermis contains a more complex pattern of keratins than thin skin and it is characterized by the constitutive expression of K6, 16, 17 and K9 [52,53]. Mutations in K6, K16 and K17 genes cause pachyonychia congenita (thick nails, plantar keratoderma) [49].
Hallmarks of activated keratinocytes include cell hypertrophy, altered cell adhesion and juxtanuclear reorganization of the keratin intermediate filament network, allowing activated keratinocytes to quickly migrate into the wound site, repopulate the skin and restore the epithelial lining and barrier function. In vitro study of mouse keratinocyte overexpressing  biopsy and collected at day3), or (C) ex vivo wounded skin were stained with K6 antibody in green or red as indicated. Non-wounded nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. In A, non-lesional skin (NL) from the same patient was used as control for PSO. In B, skin biopsy collected at day 0 was used as non-wounded control. Note that while K6 expression was not detected in all control skin epidermis, strong K6 staining was detected in the suprabasal layers of both wounded and psoriatic skin epidermis in similar patterns. Also K6 was strongly elevated in the migrating tongue of ex vivo wounded human skin at the wound edge. Details of these samples can be found in our previous published work [58].
K16 revealed that while forced expression of K16 did not alter cell proliferation, it caused a reduction in cell adhesion and K10 expression (early differentiation) [61]. K10 expression inhibits cell proliferation, but ectopic expression of K16 promotes cell proliferation and diminishes the inhibitory function of K10 on cell proliferation when K6 and K10 are co-expressed [11] . K6À/À mouse keratinocytes migrated faster than control wild-type cells [56], and K6 negatively regulates the migratory potential of skin keratinocytes by inhibiting Src kinase [62], suggesting that the migratory feature of activated keratinocytes may be regulated by an K6/ K16 independent pathway or by a non-cell autonomous manner. In contrast to K6À/À keratinocytes, K17À/À mouse keratinocytes show a delay in the closure of wounds [63], and protein translation, AKT activity and cell proliferation are suppressed in K17À/À keratinocytes [64]. These results suggest that in activated keratinocytes while the cell adhesion and differentiation maybe regulated by K6, cell hyper-proliferation and migration in response to wound maybe controlled by K17.

Regulation of K6/16/17 expression
Our group has shown that damage-associated molecular patterns "DAMPs", such as doublestranded RNA, are the first signals released from necrotic cells to rapidly initiate the inflammatory cytokine production from surrounding undamaged human keratinocyte upon injury [58,65]. Inflammatory cytokines released either from activated keratinocytes or from recruited immune cells initiate expression of hyperproliferative keratins and keratinocyte hyperproliferation. It has been shown that cytokines interleukin 1(IL1) and tumor necrosis factor α (TNFα) can synergistically induce the transcription of K6 through transcription factor C/EBPβ and NFκB [66]. Transcription factor AP1 (c-Fos + c-Jun) can also activate K6 promoter synergistically with NFκB under inflammatory conditions [19]. Additionally, transcription factor NRF2 translocated from cytoplasm to nucleus upon stimulation with proinflammatory cytokines such as IL 17 or IL 22, and upregulated the expression of K6, K16 and K17 genes via the antioxidant responsive element (ARE)-binding region, promoting proliferation of keratinocytes in psoriasis [67]. K17 expression could also be induced by IFNγ in skin epidermis, and the K17 in turn function as an auto-antigen to stimulate proliferation of T cells and IFN expression, contributing to the amplification of the autoimmune response and immunopathogenesis of psoriasis [68,69]. Accumulating recent evidences have suggested that microRNAs also play important roles in modulating keratinocyte activation and skin inflammation [70,71]. Keratin expression can be regulated by microRNA in the contact of psoriasis. For example, miR-486-3p targeted K17 mRNA for degradation and it was identified as the top downregulated microRNAs in psoriasis, leading to K17 overproduction and hyperproliferation in psoriatic keratinocytes [72]. Cutaneous squamous cell carcinoma (SCC) is the second most common skin cancers and represents about 20% of all skin cancer, with up to 700,000 new cases annually diagnosed in the USA, and it is associated with a substantial risk of metastasis [73]. SCC is characterized by extensive expression of K5/K14 through the epidermis and the expression of hyperproliferative keratin K6, K16 and K17 [50], which are not only upregulated in inflammatory skin, but often upregulated in many tumors originating in stratified and pseudostratified epithelia. K1/K10 may also be focally expressed in SCCs, and K8/K18 is often detected in poorly differentiated SCCs, and the role of K8/K18 will be discussed in more details next.

SCCs and K8/K18
As we have described in chapter 2, the simple epithelia-specific keratin pair, K8/K18, are expressed in keratinocyte progenitors, early on during embryonic skin development, and upon vertical epidermal stratification K8/K18 expression is then substituted by K5/K14, and becomes eventually lost in fully mature skin epidermis [9,10,74]. Overexpressing human K8 in mouse epidermis (TGHK8 mice) lead to severe epidermal phenotypes including epidermal hyperplasia associated with orthokeratotic hyperkeratosis, dysplastic hair follicles and altered expression terminal differentiation markers [75]. The severity of these skin phenotypes increased during aging, and the aged TGHK8 mice developed spontaneous premalignant skin tumors, and TGHK8 mice showed a drastic increase in the malignant progression of skin tumors in mouse model of chemical skin carcinogenesis. Previously, we have shown that Ctip2À/À mouse keratinocytes that overexpressed K8 exhibited loss of cell-cell contact and contain much thicker central stress fibers [6], indicating that aberrant K8 expression may decrease cell-cell adhesion and trigger an EMT (epithelial-mesenchymal transition) phenotype in epidermal keratinocytes. These results suggest that expression of K8 in adult epidermis impairs the normal epidermal differentiation program and may be responsible for the invasive behavior of transformed epidermal keratinocytes. Indeed, in adult skin epidermis, aberrant K8/K18 expression is broadly correlated with increased invasiveness and poor prognosis of squamous cell carcinomas [76]. In addition, K8/K18 protect epithelial cells against apoptosis mediated by proapoptotic signals, such as TNFα [77] released by macrophages and T lymphocytes and Fas [78], and may enable cancer cells to resist immune cell-mediated cell death and escape immune surveillance.

Regulation of keratin expression and function in SCCs
Phosphorylation is the key post-translational modification that regulates keratin functions, and phosphorylation of K8 is among the most well studied in keratin family [79]. More than a dozen phosphorylation sites have been identified on serine residues of K8 [79], and phosphorylation of K8 enhances the migratory, proliferative and invasive potential of epithelial cells, therefore promoting the malignant transformation of cancer cells [80]. Keratin expression can be altered through epigenetic modification during malignant transformation. A recent study investigating genome-wide DNA methylation changes in the progression from healthy human epidermis to cSCC reveals that DNA methylation profiles of cSCC epidermis display classical features of cancer methylomes compared to normal epidermis samples [81]. Further analyses of DNA methylation patterns of keratin gene clusters (including basal cell keratins K5 and K14 which are ectopically expressed throughout cSCC epidermis) identified major DNA methylation differences between healthy donors and cSCC patients, suggesting that abnormal keratin expression in SCCs may be regulated through epigenetic mechanism such as DNA methylation.

Conclusion
Tissue and cell differentiation specific expression of pair between type I and type II Keratins play essential roles in forming the intermediate filaments and providing cytoskeletal and structural support and mechanical resilience for epithelia tissues. In addition to these structural roles, keratins also control cell migration, cell adhesion, proliferation and differentiation processes in keratinocytes and modulate immune system in various settings. The transcriptions of keratins are tightly controlled by a series of transcription factors, and keratin functions are also regulated by post-translational modifications such as phosphorylation and intermolecular disulfide bond formation. Mutation or dysfunction of basal keratins K14/K5 or suprabasal keratins K1/K10 lead to severe skin blistering diseases, whereas K6, K16 and K17 are rapidly induced in activated keratinocytes upon skin wounding and are also expressed in inflammatory skin diseases, such as psoriasis. Understanding keratin functions and related regulatory mechanisms will help to design new therapeutic interventions for keratin-related skin diseases.