Distinct Roles for ROCK1 and ROCK2 in the Regulation of Keratinocyte Differentiation

Background

The human epidermis is comprised of several layers of specialized epithelial cells called keratinocytes. Normal homoeostasis of the epidermis requires that the balance between keratinocyte proliferation and terminal differentiation be tightly regulated. The mammalian serine/threonine kinases (ROCK1 and ROCK2) are well-characterised downstream effectors of the small GTPase RhoA. We have previously demonstrated that the RhoA/ROCK signalling pathway plays an important role in regulation of human keratinocyte proliferation and terminal differentiation. In this paper we addressed the question of which ROCK isoform was involved in regulation of keratinocyte differentiation.

Methodology and Principal Findings

We used RNAi to specifically knockdown ROCK1 or ROCK2 expression in cultured human keratinocytes. ROCK1 depletion results in decreased keratinocyte adhesion to fibronectin and an increase in terminal differentiation. Conversely, ROCK2 depletion results in increased keratinocyte adhesion to fibronectin and inhibits terminal differentiation.

Conclusion

These data suggest that ROCK1 and ROCK2 play distinct roles in regulating keratinocyte adhesion and terminal differentiation.     

The role of cathepsin E in terminal differentiation of keratinocytes

Cathepsin E (CatE) is predominantly expressed in the rapidly regenerating gastric mucosal cells and epidermal keratinocytes, in addition to the immune system cells. However, the role of CatE in these cells remains unclear. Here we report a crucial role of CatE in keratinocyte terminal differentiation. CatE deficiency in mice induces abnormal keratinocyte differentiation in the epidermis and hair follicle, characterized by the significant expansion of corium and the reduction of subcutaneous tissue and hair follicle. In a model of skin papillomas formed in three different genotypes of syngeneic mice, CatE deficiency results in significantly reduced expression and altered localization of the keratinocyte differentiation induced proteins, keratin 1 and loricrin. Involvement of CatE in the regulation of the expression of epidermal differentiation specific proteins was corroborated by in vitro studies with primary cultures of keratinocytes from the three different genotypes of mice. In wild-type keratinocytes after differentiation inducing stimuli, the CatE expression profile was compatible to those of the terminal differentiation marker genes tested. Overexpression of CatE in mice enhances the keratinocyte terminal differentiation process, whereas CatE deficiency results in delayed differentiation accompanying the reduced expression or the ectopic localization of the differentiation markers. Our findings suggest that in keratinocytes CatE is functionally linked to the expression of terminal differentiation markers, thereby regulating epidermis formation and homeostasis.     

Epidermal keratinocytes: regulation of multiple cell phenotypes by multiple protein kinase C isoforms

Squamous cells form the outermost layers of the epidermis, and though they are readily discarded from the tissue, they serve a vital water barrier function while in the stratum corneum. The generation of cornified or squamous keratinocytes involves a complex, multi-step differentiation process that insures the proper physical and immunological barrier functions of the epidermis are maintained. The regulation of keratinocyte terminal differentiation is influenced by a large number of signaling pathways. This article will review some recent findings regarding the roles of the protein kinase C (PKC) family in normal keratinocyte differentiation, as well as their involvement in skin diseases, especially skin cancer.     

Decorin gene expression and its regulation in human keratinocytes

In various cell types, including cancer cells, decorin is involved in regulation of cell attachment, migration and proliferation. In skin, decorin is seen in dermis, but not in keratinocytes. We show that decorin gene (DCN) is expressed in the cultured keratinocytes, and the protein is found in the cytoplasm of differentiating keratinocytes and in suprabasal layers of human epidermis. RT-PCR experiments showed that DCN expression is regulated by pro-inflammatory and proliferative cytokines. Our data suggest that decorin should play a significant role in keratinocyte terminal differentiation, cutaneous homeostasis and dermatological diseases.     

RIP4 is an ankyrin repeat-containing kinase essential for keratinocyte differentiation

The epidermis is a stratified, continually renewing epithelium dependent on a balance among cell proliferation, differentiation, and death for homeostasis. In normal epidermis, a mitotically active basal layer gives rise to terminally differentiating keratinocytes that migrate outward and are ultimately sloughed from the skin surface as enucleated squames. Although many proteins are known to function in maintaining epidermal homeostasis, the molecular coordination of these events is poorly understood. RIP4 is a novel RIP (receptor-interacting protein) family kinase with ankyrin repeats cloned from a keratinocyte cDNA library. RIP4 deficiency in mice results in perinatal lethality associated with abnormal epidermal differentiation. The phenotype of RIP4(-/-) mice in part resembles that of mice lacking IKKalpha, a component of a complex that regulates NF-kappaB. Despite the similar keratinocyte defects in RIP4- and IKKalpha-deficient mice, these kinases function in distinct pathways. RIP4 functions cell autonomously within the keratinocyte lineage. Unlike IKKalpha, RIP4-deficient skin fails to fully differentiate when grafted onto a normal host. Instead, abnormal hair follicle development and epidermal dysplasia, indicative of progression into a more pathologic state, are observed. Thus, RIP4 is a critical component of a novel pathway that controls keratinocyte differentiation.     

Terminal differentiation of nail matrix keratinocytes involves up-regulation of DNase1L2 but is independent of caspase-14 expression

Abstract Terminal differentiation of keratinocytes in the epidermis and in epidermal appendages results in specialized forms of cell death. Keratinocytes of the nail matrix differentiate into nail corneocytes, the building blocks of the nail plate. Here, we show that, in contrast to the abrupt breakdown of the nucleus during corneocyte formation of epidermal keratinocytes, chromatin undergoes progressive condensation over several nail matrix cell layers below the transition zone to the nail plate, where nuclear DNA disappears. Virtually all keratinocytes in the cell layer immediately beneath the nail plate contained terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling-positive DNA fragments. Nail matrix keratinocytes lacked processed caspase-3, a marker of apoptosis, and did not express caspase-14, a protease up-regulated during terminal differentiation of epidermal keratinocytes. By contrast, DNase1L2, which is also up-regulated during the differentiation of epidermal keratinocytes and plays an essential role in differentiation-associated degradation of nuclear DNA in epidermal keratinocytes, was strongly expressed in the nail matrix–nail plate transition layer. Our results show that caspase-14 is not strictly, if at all, required for differentiation-associated keratinocyte cell death and implicates DNase1L2 in terminal differentiation of nail matrix keratinocytes.     

RXR-alpha ablation in skin keratinocytes results in alopecia and epidermal alterations

RXR-alpha is the most abundant of the three retinoid X receptors (RXRs) in the epidermis. In this study, we have used Cre-mediated recombination to selectively disrupt the mouse gene for RXR-alpha in epidermal and hair follicle keratinocytes. We show that RXR-alpha is apparently dispensable for prenatal epidermal development, while it is involved in postnatal skin maturation. After the first hair pelage, mutant mice develop a progressive alopecia, histologically characterised by the destruction of hair follicle architecture and the formation of utriculi and dermal cysts in adult mice. Our results demonstrate that RXR-alpha plays a key role in anagen initiation during the hair follicle cycle. In addition, RXR-alpha ablation results in epidermal interfollicular hyperplasia with keratinocyte hyperproliferation and aberrant terminal differentiation, accompanied by an inflammatory reaction of the skin. Our data not only provide genetic evidence that RXR-alpha/VDR heterodimers play a major role in controlling hair cycling, but also suggest that additional signalling pathways mediated by RXR-alpha heterodimerised with other nuclear receptors are involved in postnatal hair follicle growth, and homeostasis of proliferation/differentiation of epidermal keratinocytes and of the skin's immune system.     

Effect of epidermal growth factor on the sheet formation of the human keratinocyte in cell culture

Epidermal growth factor is an important element in maintaining keratinocyte proliferation and maturation. To evaluate its effect on keratinocyte growth in vitro, human foreskins were cultured. The epidermal keratinocyte growth in culture was separated into two stages by a conditional medium: the proliferation stage, in which the cells were maintained in a monolayer; and the differentiation stage, in which the cells grew to stratification and keratinization. The keratinocytes were cultured in various concentrations of epidermal growth factor, and their morphology and growth behavior were closely observed. Our results demonstrated that cultured keratinocytes grew in a confluent layer under the influence of epidermal growth factor. In contrast, in a culture without epidermal growth factor, the proliferation rate of cultured keratinocytes slowed down and eventually the cells stopped growing. During serum stimulation, with or without additional exogenous epidermal growth factor, the cultured keratinocytes grew continuously to the normal terminal differentiation. Under this two-stage culture model, the cultured keratinocytes could grow into an intact sheet of graftable epidermis.     

Temporal and spatial sequence expression of cytokeratin K19 in cultured human keratinocyte

The unique cytokeratin K19 specifically expresses in simple epithelial cells, basal cells of non-keratinized stratified squamous epithelium, epidermal cells during the embryonic stage and squamous carcinoma cells, but it is not expressed in adult epidermis. Interestingly, when epidermal cells are cultured in vitro, K19 is re-expressed in the supra-basal layer. K19 expression was used as a marker for epidermal cell growth and differentiation. In order to clarify the temporal and spatial sequential expression in cultured keratinocyte, two-stage human keratinocyte culture systems were used to examine K19 expression in keratinocytes in a proliferation and differentiation stages through immunoblotting and immunohistochemistry assay. According to our results, K19 was not expressed in cultured human keratinocytes in the proliferation stage but was re-expressed in keratinocytes three days after the cultured medium was changed to a differentiation medium. Immunohistochemical observation revealed that K19 was persistently expressed in the supra-basal layer of cultured keratinocytes during first three weeks of culturing, but none was detectable in the basal cell layer. When keratinocytes were cultured with an "inserted cultured dish," K19 was persistently expressed in all layers of keratinocytes nourished by medium both from an inner chamber and an outer chamber. The different expression of K19 in these two different culture systems seemed to indicate that down regulation of K19 expression in keratinocyte was related to the direction of medium supply.     

PKC�� promotes a proliferation to differentiation switch in keratinocytes via upregulation of p27Kip1 mRNA through suppression of JNK/c-Jun signaling under stress conditions

To maintain epidermal homeostasis, the balance between keratinocyte proliferation and differentiation is tightly controlled. However, the molecular mechanisms underlying this balance remain unclear. In 3D organotypic coculture with mouse keratinocytes and fibroblasts, the thickness of stratified cell layers was prolonged, and growth arrest and terminal differentiation were delayed when PKCη-null keratinocytes were used. Re-expression of PKCη in PKCη-null keratinocytes restored stratified cell layer thickness, growth arrest and terminal differentiation. We show that in 3D cocultured PKCη-null keratinocytes, p27^Kip1 mRNA was downregulated, whereas JNK/c-Jun signaling was enhanced. Furthermore, inhibition of JNK/c-Jun signaling in PKCη-null keratinocytes led to upregulation of p27^Kip1 mRNA, and to thinner stratified cell layers. Collectively, our findings indicate that PKCη upregulates p27^Kip1 mRNA through suppression of JNK/c-Jun signaling. This results in promoting a proliferation to differentiation switch in keratinocytes.     

Epidermal expression of the full-length extracellular calcium-sensing receptor is required for normal keratinocyte differentiation

The importance of the extracellular calcium-sensing receptor (CaR) in the stringent control of extracellular Ca(2+) concentration is well established. However, the presence of CaR in tissues not directly involved in regulating mineral ion homeostasis such as the epidermis suggests a role for CaR in other cellular functions. Although extracellular Ca(2+) regulates the differentiation of epidermal keratinocytes, the role of CaR in this process in the epidermis is not fully understood. In this study we showed using in situ hybridization and immunohistochemistry that CaR is expressed in suprabasal keratinocytes of the mammalian epidermis. We then evaluated the changes in epidermal keratinocyte morphology and differentiation in Casr(-/-) mice lacking the full-length CaR. These mice show increased expression of an alternatively spliced form of CaR which lacks acute Ca(2+)-signaling properties. The absence of the full-length CaR in the epidermis resulted in ultrastructural changes (abnormal keratohyalin granule formation and precocious lamellar body secretion) in the terminally differentiated granular keratinocytes. Furthermore, the expression of both mRNA and protein for the calcium inducible keratinocyte differentiation markers, filaggrin and loricrin, were down-regulated in the epidermis of Casr(-/-) mice, whereas the number of proliferating cells were increased even though the calcium gradient within the epidermis was enhanced. Our results demonstrate that the epidermal expression of the full-length CaR is required for the normal terminal differentiation of keratinocytes.     

Stratum corneum-derived caspase-14 is catalytically active

Caspase-14, a cysteine protease with restricted tissue distribution, is highly expressed in differentiated epidermal keratinocytes. Here, we extracted soluble proteins from stratum corneum (SC) of human epidermis and demonstrate that the extract cleaves tetrapeptide caspase substrates. The activity decreased to below 10% when caspase-14 was removed by immunodepletion showing that caspase-14 is the predominant caspase in SC. In contrast to normal SC, where caspase-14 was present exclusively in its processed form, incompletely matured SC of parakeratotic skin from psoriasis and seborrheic dermatitis contained both procaspase-14 and caspase-14 subunits. Fractionation of extract from parakeratotic SC revealed that the peak caspase activity coeluted with processed caspase-14 but not with procaspase-14. Our results suggest that during regular terminal keratinocyte differentiation, endogenous procaspase-14 is converted to caspase-14 subunits that are catalytically active in the outermost layers of normal human skin.     

Ligand Targeting of EphA2 Enhances Keratinocyte Adhesion and Differentiation via Desmoglein 1

EphA2 is a receptor tyrosine kinase that is engaged and activated by membrane-linked ephrin-A ligands residing on adjacent cell surfaces. Ligand targeting of EphA2 has been implicated in epithelial growth regulation by inhibiting the extracellular signal-regulated kinase 1/2 (Erk1/2)-mitogen activated protein kinase (MAPK) pathway. Although contact-dependent EphA2 activation was required for dampening Erk1/2-MAPK signaling after a calcium switch in primary human epidermal keratinocytes, the loss of this receptor did not prevent exit from the cell cycle. Incubating keratinocytes with a soluble ephrin-A1-Fc peptide mimetic to target EphA2 further increased receptor activation leading to its down-regulation. Moreover, soluble ligand targeting of EphA2 restricted the lateral expansion of epidermal cell colonies without limiting proliferation in these primary cultures. Rather, ephrin-A1-Fc peptide treatment promoted epidermal cell colony compaction and stratification in a manner that was associated with increased keratinocyte differentiation. The ligand-dependent increase in keratinocyte adhesion and differentiation relied largely upon the up-regulation of desmoglein 1, a desmosomal cadherin that maintains the integrity and differentiated state of suprabasal keratinocytes in the epidermis. These data suggest that keratinocytes expressing EphA2 in the basal layer may respond to ephrin-A1–based cues from their neighbors to facilitate entry into a terminal differentiation pathway.