A question that still challenges cell and tissue biologists is that of the driving forces that have selected for and conserved the numerous intermediate filament proteins in vertebrates. We are only beginning to understand the functions of this family of cytoskeleton structures and we have very few experimental tools for testing comparative functions of these proteins in a satisfactory way. A major turning point came with the discoveries that mutations in keratin intermediate filament genes were responsible for a large number of inherited skin fragility disorders. These disease links showed unequivocally that intermediate filament proteins, at least in barrier epithelia like skin, provide essential stress resilience for cells in tissues. The keratin genes account for three quarters of all the intermediate filament genes identified in the human genome, and it seems highly likely that any function attributable these structural proteins will also be important for the nonkeratin intermediate filaments. Once the stress resistance function of intermediate filaments is taken as a fact, rather than a persistent speculation, this knowledge can guide further experimental analysis and design to allow us to at last move closer to understanding the biology of these enigmatic cytoskeleton filaments.

Epidermal Blistering Caused by Keratin Mutations

The idea that keratins may contribute a structural supporting role in epithelia shifted from hypothesis to well-accepted fact when it was discovered that mutations in keratins K5 and K14 caused profound fragility of epidermal basal keratinocytes. Analyses of a wide range of heritable skin disorders described below have confirmed that without keratin intermediate filaments to form an effective network in the cytoplasm of keratinocytes, these normally very tough epithelial cells are rendered fragile and break down when the skin is subjected to quite mild everyday physical stress, such as stretching or rubbing. As cells expressing mutant keratins break down, they destroy cell layers in the stratified epithelium.

The first of these to be identified as caused by keratin mutations was a group of disorders known as epidermolysis bullosa simplex (EBS), with bullosa referring to the bullous or fluid-filled blisters that develop on the skin following quite mild physical trauma such as scratching or rubbing. In this case the keratinocytes of the epidermal basal layer in the skin are rendered fragile by a mutation (usually dominant) in either K5 or K14,^1-3 i.e., the pair of keratins that make up the keratin cytoskeleton of these cells. The fracture of the basal cells can be seen by histology of rubbed skin and fluid accumulates in the plane of rupture. In severe cases, the Dowling-Meara type of EBS (OMIM 131760), characteristic aggregates of electron-dense material can be (variably) seen in the cytoplasm of basal cells.

The published data on mutations in keratins, and other intermediate filaments, is collected in a public internet-accessible database (www.interfil.org) maintained here at the University of Dundee. Analysis of nearly two hundred mutations in K5 and K14 to date has identified mutations in about 78% of cases diagnosed as EBS. Nearly all the identified pathogenic mutations in these genes are dominant. There are a few cases of recessive mutations which nearly all lead to loss of function, or a natural knock-out, in K14.^4-6 No K5 knock-out mutations have been identified in humans, suggesting that this is lethal, presumably because there is no “back-up” type II keratin for K5 in basal cells. (For type I keratins, there are other possible candidates to reinforce a K14 knockout, such as K15.⁷)

The severest cases of EBS are mostly associated with mutation in a specific codon for an arginine at position 125 in the amino acid sequence of K14, as first described by Coulombe et al;⁸ otherwise they are nearly all in the helix initiation motif or the helix termination motif, the rod ends that are so critical for intermediate filament assembly. Milder forms (so-called Weber-Cockayne type EBS (OMIM 131800), and to a certain extent the Köbner type (OMIM 131900)) are associated with mutation cluster sites outside the helix boundary peptides^9,10 but they almost certainly define other critical points in the intermediate filament proteins as the pathogenic mutations are not randomly distributed (discussed in ref. 11). Overall, the severity of a case of K5/K14 EBS depends on the position of the mutation in the keratin protein.

Candidate Genes in Other Epithelia

Once the connection between K5/K14 mutations and EBS was made, there followed a period of intense analysis of other inherited dermatological conditions whose phenotype suggested cell fragility. Amongst other benefits, this period undoubtedly brought dermatologists and basic scientists together in an interesting and highly productive way. Many more EBS mutations were found in K5 and/or K14, although as they were mostly sought only in the helix boundary regions (following the first findings) it is probable that many were missed at first. Current good practice in EBS screening must involve sequencing the whole coding region of these two genes.

It was quickly realised that other phenotypically diverse skin conditions might also be caused by mutations in (different) keratin genes. These other disorders exhibited quite distinct phenotypes from that of EBS, yet the similarities and differences of these keratin disorders all fell rapidly into place as it was seen that the diverse phenotypes depended on the tissue expression of the mutated keratin target genes. A large amount of immunohistological information on keratin expression in tissues had been gathered since monoclonal antibodies to keratins were first generated in the early 1980s, and using this information it was now possible to rapidly identify candidate genes for other epithelia fragility disorders. Mutations were soon identified by sequence analysis of these other keratin genes. Again, most transpired to be dominant negative missense mutations situated in the helix boundary peptides of keratin genes, as had been the case for K5 and K14. This enlarging group of diseases and their relationship to keratin mutations has been the subject of a number of recent reviews.^12,13 Examples of some of the mutation reports are cited below but a more extensive list of published mutations is collated in the Intermediate Filament Database (http://www.interfil.org).

The second pair of keratins in which pathogenic mutations were identified in skin fragility disorders was the epidermal secondary or differentiation -specific pair of K1 and K10, normally expressed in suprabasal post-mitotic keratinocytes as they leave the basal layer and begin their progress towards complete terminal differentiation in the epidermis. It was correctly predicted that mutations in K1 and K10 might be responsible, at least in part, for the epithelial cell breakdown seen in the epidermolytic hyperkeratosis that characterised another group of disorders, know as bullous congenital ichthyosiform erythroderma (BCIE; OMIM 113800), also sometimes called after the characteristic epidermolytic hyperkeratosis seen in these disorders (EHK) (see http://www.interfil.org for identified mutations). With this condition, infants can sometimes be initially misdiagnosed as EBS, but the early blistering subsides to be replaced by thick, ichthyotic skin, which can be very extensive over the body surface. Histology of such skin shows that the basal cells are intact but the suprabasal cells are fractured, which by analogy with the basal cell lysis of EBS would suggest a defect in one of the pair of suprabasal keratins, i.e., K1 or K10. This proved indeed to be the case, as several groups have confirmed with the identification of a range of dominant mutations clustering around the helix boundary motifs of either of the two proteins.¹⁴ Mutations in K1 have also since been linked to epidermolytic palmo-plantar hyperkeratosis (EPPK; OMIM 144200), mild diffuse cases of nonepidermolytic palmoplantar keratosis (NEPPK; OMIM 600962)¹⁵ and possibly a case of Curth-Macklin ichthyosis hystrix (OMIM 146590).¹⁶ EPPK is more usually linked to K9 mutations;^17-19 K9 is a keratin expressed specifically in palmoplantar epidermis.

The same reasoning was applied to ichthyosis bullosa of Siemens (OMIM 146800), an uncommon epidermal fragility disorder characterised by cytolysis of upper layers of the epidermis and the formation of flat, superficial blisters. This was found to be caused by mutations in K2e,^20-22 which Franke's group had shown was a late-expressed keratin in the epidermis.²³ Thus the fragile cells again matched the known expression range of the keratin concerned.

Mutations in another pair of secondary (differentiation-specific) keratins were discovered to cause another skin fragility disorder when mutations in K4 and K13 were identified in families affected with white sponge naevus (WSN; OMIM 193900).^24-27 Loose white plaques in the buccal epithelium characterise this benign condition, which is more usually picked up by dentists than doctors. Buccal epithelium, like other regions of oro-genital epithelia, expresses K4 and K13 as the suprabasal keratin pair and WSN was found to be specifically associated with dominant mutations in these two keratins, mostly located in the helix termination motifs. Histological sections at first show a loose basketweave structure of the suprabasal cells layers, which on closer inspections can be seen to be caused by cytolysis of these cell layers.

Corneal epithelium specifically expresses keratins K3 and K12, and these two keratins have been found to harbour helix termination motif mutations in patients affected by a disorder called Meesman corneal dystrophy (MCD; OMIM 122100).²⁸ People suffering from MCD develop small blisters specifically on the corneal epithelium.

The most divergent phenotypes of keratin disorders lie in the pachyonychia congenita disorders. Pachyonychia (thick nails) congenita has two clinically distinct forms: the Jadassohn-Lewandowsky type (PC-1; OMIM 167200) with thick nails, oral leukoplakia and nonepidermolytic palmoplantar keratoderma (NEPPK; OMIM 600962), and the Jackson-Lawler type (PC-2; OMIM 167210) with thick nails, pilosebaceous cysts, pili torti (twisted or corkscrew hairs) and, variably, prematurely erupted natal teeth. Guided by candidate gene identification based on a knowledge of tissue expression patterns of keratin, PC-1 was linked with mutations in K6a or K16, and PC-2 with mutations in K6b and K17.^29-32 Milder mutations in K6b or K17 are associated with steatocystoma mutiplex (OMIM 184500) suggesting that there is a severity spectrum in these genes analogous to that seen in EBS.^32,33

There are also mutations in some hair keratin genes that are associated with hair fibre malformations such as monilethrix (OMIM 158000),^34-36 which may also be explainable by cell fragility in a critical subpopulation of cells involved in shaping the forming hair. In all these disorders the predominant location of the mutations has been found to be in the helix boundary motifs, and sometimes in the H1 domain of the head region adjacent to the N-terminal end of the rod domain. It is only in the K5/K14 mutations of EBS that a much wider range of mutation clusters along the proteins is seen. This is most likely because mutated secondary keratins are to some extent protected or reinforced by the underlying template of residual K5/K14 filaments synthesised in the basal cells and serving as an assembly template for the secondary keratins. Thus only the most disruptive type of mutations are pathogenic in the secondary keratins.

Most of the keratin disorders show skin thickening (keratoderma), especially on the palms and soles (palmoplantar skin). This is probably a result of the “chronic wounding” of cell fragmentation: cells breaking open will release cytokines to trigger a repair process, even though the epithelial barrier is not completely breached. Cell proliferation in epidermal wounding is normally part of the later phase of epidermal response, with the earlier epithelial response directed to cell migration to close the breech in the barrier. Thus the proliferative response may not need to detect free space around the cells whereas a migration response would.

From Candidate Genes to “Candidate Diseases”

For several years, there were notable omissions from the list of keratins implicated in epithelial fragility disorders, in the simple epithelial keratins such as K8/K18 (the primary keratins) and K7, K19 and K20. This might suggest that simple epithelial keratins were not so essential for cell resilience and survival as they mostly occur in internal epithelia that are not so greatly stressed as epidermal tissues. On the other hand, K8/K18 are expressed in the very earliest stages of embryogenesis, and embryos of homozygous knockout mice have greatly reduced viability.³⁷ The tissue expression patterns of K8/K18 suggested to us that intestinal epithelia might be vulnerable to K8/K18 mutation-induced fragility and so we examined DNA from patients with inflammatory bowel disease. Mutations have now been identified in some cases of cryptogenic liver cirrhosis (included in OMIM 215600),³⁸ pancreatitis³⁹ and in our case from inflammatory bowel disease—both Crohn disease (OMIM 266600) and ulcerative colitis (OMIM 191390).⁴⁰ Strikingly these mutations are not in the helix boundary motifs, suggesting that mutations at such critical sites might be embryonic lethal in these early-expressed keratin genes. However neither are the mutations dominant; they occur in polygenic disorders, so at best they can only be predisposing factors. This is a good example of the necessity for lateral thinking about possible consequences of cytoskeleton defects in tissues.

Supramolecular Stability versus Molecular Flux

So how does this information sit with our prior understanding of keratin function in cells? Intermediate filaments are still far less well understood than the other cytoskeleton filamentous polymer systems of actin and tubulin. In contrast with actin and tubulin, intermediate filaments have been widely believed (somewhat erroneously) to be inert and unreactive. Intermediate filaments appear so because they form complex and often very dense meshworks of rope-like polymers in the cytoplasm that are not easily disrupted by drugs, nor easily soluble. Moreover the filaments are multi-stranded and have no net polarity, so dynamic flux is much more difficult to detect and monitor than in actin filaments and microtubules. Purified intermediate filament proteins in vitro will polymerise rapidly without any requirement for cofactors or associated proteins: if this is also the case inside cells, this only further increases the difficulty of analysing single strand kinetics.

In recent years several lines of evidence have accumulated which suggest that cytoplasmic intermediate filaments are much more dynamic than was previously thought. The increasing use of techniques such as green fluorescent protein (GFP), for example, to tag and track specific proteins in living cells has revealed that much subunit exchange takes place along filaments. ⁴¹ The speed of this flux reflects the likelihood that subunits of protein are normally flickering on and off the filaments all along their length,⁴² reviewed by Helfand et al.⁴³ Small nonfilamentous protein particles of vimentin⁴⁴ and keratin⁴⁵ have been observed that are highly mobile and that associate with actin and tubulin polymers as well as intermediate filaments. Larger nonfilamentous aggregates accumulate in some epithelial cells during mitosis^46,47 and can be induced experimentally by intracellular injection of some antibodies.^48-50 In some diseases, nonfilamentous keratin aggregates accumulate spontaneously, such as in severe forms of the genetic skin blistering condition epidermolysis bullosa simplex⁵¹ and in hepatocytes of people with alcoholic cirrhosis of the liver (Mallory bodies).⁵²

Keratin aggregates in many forms are associated with a high level of phosphorylation^53-55 and elevated phosphorylation of intermediate filaments is associated with the depolymerised state, suggesting that filaments are remodelled inside cells by cycles of phosphorylation and dephosphorylation.^56-58 Specific information on phosphorylation sites and their regulating enzymes is still patchy for intermediate filaments and is best defined so far for the simple epithelial keratins K8 and K18 (see Owens and Lane⁵⁹ for a recent review).

Thus, intermediate filaments such as keratins clearly have mechanisms in place that can facilitate rapid spatial modulation at many levels, from subunit flux along the filament surface to total filament collapse and remodelling by phosphorylation. Their apparent stability is probably due to the multistranded structure of the filament bundles seen by light microscopy which protects these cytoskeleton structures from total catastrophic collapse as can take place in microtubules and actin filaments. Thus, under most circumstances the tonofilament as a whole persists. The evolution of a system with this supramolecular stability indicates that network persistence is an important aspect of intermediate filament function. Finally, the recent observation that accumulation of keratin aggregates in EBS-derived keratinocytes is exacerbated by mechanical stress⁶⁰ indicates that keratin filament remodelling can take place as a direct response to mechanical stress, thus putting all the pieces in place for keratins to function as mechanical stress sensors in epithelia.

Keratin Filaments As Tensile Structures

Keratins are the intermediate filament proteins characteristic of epithelial sheet tissues and account for up to 80% of the total cell protein in differentiated keratinocytes. A typical immunofluorescence image of keratin filaments (fig. 1) shows a dense mesh of cytoplasmic fibres running in all directions through the cell in bundles of varying thickness, linked into desmosomes at cell to cell junctions and to hemidesmosomes at the cell-substrate interface. Keratin staining images from cells in culture and in tissue sections all show evidence of trans-tissue continuity of keratin filament bundles (tonofilaments) via cell junctions.

Figure 1

Immunofluorescence image of confluent keratinocytes stained with antibody to keratin, illustrating the radial tonofilaments that extend from the cell periphery into the cell body. They are typically in direct alignment with filaments subtending the other (more...)

Do these keratin filaments sustain tension? The old term “tonofilaments” for keratin filament bundles presumes a degree of tension in the system, probably suggested by the straight arrangement of peripheral radial keratin bundles that subtend the desmosomes in keratinocytes (see fig. 1). This first impression probably merits closer inspection as if there is tension within the keratin cytoskeleton of an epithelial cell, it is probably not evenly distributed. Seen with fluorescence light microscopy, the peripheral radial filament bundles appear run end-on into desmosomes (although by electron microscopy they appear to loop through the plaques) and are in directional alignment with their reciprocal fibres on the other side of the desmosomes in the neighbouring linked cell, suggesting a pull on both sides of a desmosome.

Further back into the cell body, these straight tonofilaments mostly become subsumed into a more ring-shaped arrangement of filaments that encircle the nucleus in the plane of the substrate. This suggests that any tension on the desmosomes must now have been dissipated or redistributed between a different set of vectors. Time-lapse filming of GFP-keratins in living cells shows sinuous wave-like flexing of keratin bundles,⁴¹ indicating a reduced tension in at least some parts of the system. Electron microscopic images suggest that the filaments do not end at the junctions but loop through the desmosomes/hemidesmosome plaques.⁶¹ This would intuitively seem to be functionally important, as it suggests side-on associations of filaments with junction proteins, i.e., associations involving sites that repeat along the filaments. This would allow slippage of filaments through or past the anchorage junctions and so preventing rupture of structures by mechanical stress in the epithelial sheet. Thus the cytoplasmic distribution of keratin tonofilaments suggests that some locally variable tension is maintained in the system, and the evidence for dynamic turnover suggests that filaments can be repositioned, possibly in response to mechanical cues, providing the basis for a mechanosensory system based on intermediate filaments.

Significance of Keratin Role in Skin Fragility Disorders

Disease associations have now been identified for 15 of the 21 well-studied keratins expressed in soft epithelial cells. Those not so far associated with pathogenic mutations are K2p, K7, K15, K19 and K20. K15 seems to be a secondary basal cell keratin in keratinocytes, associated with a long-term stability state, and K7, K19 and K20 are all secondary simple epithelial keratins. At least 4 other nonkeratin intermediate filaments have been associated with diverse pathologies from premature ageing (lamin mutations: Hutchinson-Gilford progeria (OMIM 176670), Emery-Dreifuss muscular dystrophy (OMIM 181350) and familial partial lipodystrophy, OMIM 151660), to desmin mutations in myofibrillar myopathy (OMIM 601419) and GFAP mutations in Alexander disease (OMIM 203450) and neurofilament mutations in some cases of ALS (OMIM 105400) (reviewed in Lane and Pekny, 2004).

The hypothesis that keratins have a role in maintaining mechanical resilience in epithelia arises very naturally from observing the configuration of keratin filament bundles (tonofilaments) and desmosome cell-cell junctions by immunofluorescence microscopy. Until the discovery of keratin mutations in skin disorders, there had been no way of proving it. Keratins are hard to get rid of: in cultured cells they appear to be refractory to most of the drugs used to disrupt actin and tubulin systems, and even microinjection of antibodies, which can be very effective in specifically disrupting keratins as we showed earlier,^48,62 did not destroy epithelial cells in tissue culture. This is probably because there is no significant mechanical stress involved in cells attached and spread on the stiff plastic substrate of a tissue culture dish.

The keratin mutations clearly show that cell fragility arises from loss of cell resilience , due to loss of function of the keratin cytoskeleton. With the filament system compromised by certain types of mutations in one of the relevant keratin genes, the cell's internal reinforcement is inadequate and the cell is torn apart by mechanical forces acting on the skin that would normally cause no detectable response in the tissue. The mechanisms for the nonkeratin disorders seem more obscure at first sight. However it seems likely that these too will eventually be recognized as resulting from structural fragility. More intermediate filament links with diseases will continue to slowly emerge as we become better at predicting the consequences of intermediate filament mutations and as large-scale sequencing becomes more trivial.

Therefore, besides the not-insignificant benefit of being able to inform patients about their disease, and to carry out prenatal screening where appropriate and where required, what else can we learn from these disorders? We should start to consider the downstream consequences of the findings in human skin diseases.

First, the disease associations show beyond doubt that keratin intermediate filament proteins are required to provide essential resilience to cells. Thus this concept of intermediate filament function has now progressed from the status of a persistent but logical speculation to that of a text-book fact. Armed with this fact, we can begin to progress to the next paradigm level of understanding intermediate filaments, such as querying the biological need for different keratins in different tissues, and from there to probe the likely evolutionary benefit of so many different intermediate filament genes. The simplest explanation for why vertebrates need different stress-resisting intermediate filaments in different tissues is that the filament proteins are subtly different and that the fine-tuning of the filament cytoskeleton in different tissues has evolved to match different tissue cell requirements.

Secondly, if it is accepted that different intermediate filaments impart different resilience characteristics to cells of different tissues, and that this stress resilience is essential for tissue function, then it is likely that the ability to sense mechanical stress and initiate mechanical stress-specific signal cascades in tissues, especially epithelia, is also an important feature of all tissues expressing specific intermediate filaments. This is a concept that has received very little attention to date except in specialised tissues like muscle. It is now clear that keratins and other intermediate filaments have mechanisms in the cell for rapid remodelling, and that this remodelling can be triggered by mechanical stress such as cell deformation by stretching⁶⁰ or swelling;⁶³ stress remodelling of intermediate filament has been seen in other cell types too.^64,65 All the mechanisms are in place for intermediate filaments to function as stress sensors in tissue cells; the signal transduction pathways triggered by biological tissue deformation events now need to be identified.

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