Opposite Effects of Ras or PKC Activation on the Expression of the SPRR2A Keratinocyte Terminal Differentiation Marker

Epidermal growth factor (EGF) enhances the expression of the keratinocyte terminal differentiation marker SPRR2A, when added to monolayers of basal keratinocytes, induced to stratify by increasing the extracellular calcium concentration. A similar stimulation is found during suspension-induced differentiation in methylcellulose. This effect, which is observed after several hours of EGF addition, is restricted to terminally differentiating keratinocytes and is dependent on PKC signaling. EGF also transiently activates the Ras signaling pathway, with a maximum induction after 10 min (Medema et al., 1994, Mol. Cell. Biol. 14, 7078-7085). The cellular effects of activated Ras were determined by transient transfection of Ha-ras(Leu-61) into normal human keratinocytes. Activated Ras completely inhibited PKC-mediated expression of SPRR2A. This inhibition is mediated via c-Jun as it is reversed by a dominant-negative c-Jun mutant (cJunDelta6/194) and c-Jun can substitute for activated Ras. The inhibitory effect is targeted to a 150-bp minimal promoter region, which is essential and sufficient for SPRR2A expression during keratinocyte terminal differentiation. This indicates that the Ras and PKC pathways, which both can be triggered by EGF, although at different time points, have opposite effects on SPRR2A gene expression.     

Structural organization and regulation of the small proline-rich family of cornified envelope precursors suggest a role in adaptive barrier function

The protective barrier provided by stratified squamous epithelia relies on the cornified cell envelope (CE), a structure synthesized at late stages of keratinocyte differentiation. It is composed of structural proteins, including involucrin, loricrin, and the small proline-rich (SPRR) proteins, all encoded by genes localized at human chromosome 1q21. The genetic characterization of the SPRR locus reveals that the various members of this multigene family can be classified into two distinct groups with separate evolutionary histories. Whereas group 1 genes have diverged in protein structure and are composed of three different classes (SPRR1 (2x), SPRR3, and SPRR4), an active process of gene conversion has counteracted diversification of the protein sequences of group 2 genes (SPRR2 class, seven genes). Contrasting with this homogenization process, all individual members of the SPRR gene family show specific in vivo and in vitro expression patterns and react selectively to UV irradiation. Apparently, creation of regulatory rather than structural diversity has been the driving force behind the evolution of the SPRR gene family. Differential regulation of highly homologous genes underlines the importance of SPRR protein dosage in providing optimal barrier function to different epithelia, while allowing adaptation to diverse external insults.     

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.     

ZNF750 Is Expressed in Differentiated Keratinocytes and Regulates Epidermal Late Differentiation Genes

Disrupted skin barrier due to altered keratinocyte differentiation is common in pathologic conditions such as atopic dermatitis, ichthyosis and psoriasis. However, the molecular cascades governing keratinocyte terminal differentiation are poorly understood. We have previously demonstrated that a dominant mutation in ZNF750 leads to a clinical phenotype reminiscent of psoriasis and seborrheic dermatitis. Here we show that ZNF750 is a nuclear protein bearing a functional C-terminal nuclear localization signal. ZNF750 was specifically expressed in the epidermal suprabasal layers and its expression was augmented during differentiation, both in human skin and in-vitro, peaking in the granular layer. Silencing of ZNF750 in Ca2+-induced HaCaT keratinocytes led to morphologically apparent arrest in the progression of late differentiation, as well as diminished apoptosis and sustained proliferation. ZNF750 knockdown cells presented with markedly reduced expression of epidermal late differentiation markers, including gene subsets of epidermal differentiation complex and skin barrier formation such as FLG, LOR, SPINK5, ALOX12B and DSG1, known to be mutated in various human skin diseases. Furthermore, overexpression of ZNF750 in undifferentiated cells induced terminal differentiation genes. Thus, ZNF750 is a regulator of keratinocyte terminal differentiation and with its downstream targets can serve in future elucidation of therapeutics for common diseases of skin barrier.     

A tale of terminal differentiation: IKKα, the master keratinocyte regulator

Keratinocyte differentiation is the process of cellular maturation from a mitotic state to a terminally differentiated state during which skin builds up a tough yet soft skin barrier to protect the body. Its irreversibility also allows the shedding of excessive keratinocytes, thereby maintaining skin homeostasis and preventing skin diseases. Although the entire journey of keratinocyte differentiation is intricate and not well understood, it is known that Ras is able to block keratinocyte terminal differentiation and instead induce keratinocyte proliferation and transformation. It appears that uncontrolled proliferation actually interrupts differentiation.

However, it has been unclear whether there are any innate surveillants that would be able to induce terminal differentiation by antagonizing excessive mitotic activities. Inhibitor of nuclear factor κB kinase-α (IKKα, previously known as Chuk) emerges as a master regulator in the coordinative control of keratinocyte differentiation and proliferation and as a major tumor suppressor in human and mouse skin squamous cell carcinomas. IKKα does so largely by integrating into the epidermal growth factor receptor (EGFR)/Ras/extracellular signal-regulated kinase (Erk)/EGFR ligand pathways during mitosis and differentiation. We discuss these findings herein to extend our understanding of how IKKα-mediated terminal differentiation serves as an innate surveillant in skin.     

Human Calmodulin-like Protein Is an Epithelial-Specific Protein Regulated during Keratinocyte Differentiation

Human calmodulin-like protein (CLP) is a calcium-binding protein down-regulated in a cell culture model of mammary tumorigenesis as well as in a majority of breast cancers in vivo. CLP down-regulation may be a result of the poorly differentiated state of these cell lines and tumors, or CLP expression may be incompatible with the uncontrolled cell growth associated with tumorigenesis. To learn more about CLP expression and regulation, we determined the distribution of CLP in various human tissues by immunohistochemistry. CLP was expressed exclusively in the epithelium of the tissues surveyed and was most abundant in thyroid, breast, prostate, kidney, and skin. CLP expression appears to increase in stratified epithelium during differentiation, as illustrated in the skin where CLP staining intensified from the basal through the spinous to the granular layers. Using a normal human keratinocyte culture model, we examined CLP expression in response to various agents known to affect keratinocyte differentiation. Agents that inhibit (epidermal growth factor, EGF) or permit (keratinocyte growth factor) terminal differentiation correspondingly regulate CLP expression. Factors modulating the EGF receptor signaling pathway were particularly potent in regulating CLP expression. CLP expression correlated with an agent's ability to promote terminal differentiation regardless of the agent's effect on keratinocyte proliferation. These studies show that CLP expression is coordinately regulated by, and may be involved in, the program of terminal differentiation in human keratinocytes and, likely, other differentiating epithelial cell types.     

Pannexin1 and Pannexin3 Exhibit Distinct Localization Patterns in Human Skin Appendages and are Regulated during Keratinocyte Differentiation and Carcinogenesis

Having shown that Panx1 and Panx3 are expressed in the epidermis, we investigated their distribution in human skin adnexal structures and skin cancer. Both proteins were found in hair follicles, sebaceous and eccrine glands, as well as blood vessels. Panx1 was detected as punctate or diffuse intracellular labeling, while Panx3 was only observed as diffuse intracellular staining, suggesting different functions. We also identified the Panx3 immunoreactive ～70 kD species modulated during keratinocyte differentiation as Panx3. Since our data indicate that pannexins are regulated during keratinocyte differentiation, we assessed whether their levels are altered under circumstances in which keratinocyte differentiation is compromised. We found that Panx1 and Panx3 levels are highly reduced in human keratinocyte tumors, thus showing for the first time that both pannexins are dysregulated in human cancers. Altogether, these data suggest that Panx1 and Panx3 have distinct and unique functions within the skin in health and disease.     

Pannexin1 and Pannexin3 Exhibit Distinct Localization Patterns in Human Skin Appendages and are Regulated during Keratinocyte Differentiation and Carcinogenesis

Having shown that Panx1 and Panx3 are expressed in the epidermis, we investigated their distribution in human skin adnexal structures and skin cancer. Both proteins were found in hair follicles, sebaceous and eccrine glands, as well as blood vessels. Panx1 was detected as punctate or diffuse intracellular labeling, while Panx3 was only observed as diffuse intracellular staining, suggesting different functions. We also identified the Panx3 immunoreactive ～70 kD species modulated during keratinocyte differentiation as Panx3. Since our data indicate that pannexins are regulated during keratinocyte differentiation, we assessed whether their levels are altered under circumstances in which keratinocyte differentiation is compromised. We found that Panx1 and Panx3 levels are highly reduced in human keratinocyte tumors, thus showing for the first time that both pannexins are dysregulated in human cancers. Altogether, these data suggest that Panx1 and Panx3 have distinct and unique functions within the skin in health and disease.     

SPRR1B overexpression enhances entry of cells into the G0 phase of the cell cycle

Many studies have established the role of SPRR1B during squamous differentiation of skin and respiratory epithelial cells. However, its role in nonsquamous cells is largely unknown. We reported that expression of SPRR1B in Chinese hamster ovary (CHO) cells is increased as they enter the G0 phase of the cell cycle. The purpose of this study was to further investigate the SPRR1B expression pattern in nonsquamous tumors and to study its role in these cells. Expression of SPRR1B was detected by Northern blotting in a higher percentage of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced compared with beryllium metal-induced rat lung adenocarcinomas. In situ hybridizations confirmed that SPRR1B is expressed in individual or clusters of cells of nonsquamous cells from mouse, rat, and human adenocarcinomas. The same pattern of expression was observed in adenocarcinomas formed in nude mice from cell lines established from adenocarcinomas. SPRR1B expression was downregulated in the cell lines derived from adenocarcinoma when cells were enriched in G0 at low confluence. Tetraploidy was induced in CHO, mouse, and human tumor cell lines by stably overexpressing SPRR1B, whereas control cells showed no change in ploidy. Inducible expression of this protein for shorter periods using the ecdyson system did not affect growth rate or the ploidy of CHO cells but accelerated entry into G0/G1 compared with controls. These findings indicate that SPRR1B is likely coupled primarily to signals responsible for withdrawal from the proliferative state rather than the final stages of cellular quiescence and that its overexpression for prolonged periods may disrupt the normal progression of mitosis.     

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.     

Molecular Characterization and Evolution of the SPRR Family of Keratinocyte Differentiation Markers Encoding Small Proline-Rich Proteins

SPRR genes (formerly SPR) encode a novel class of polypeptides (small pr oline rich proteins) that are strongly induced during differentiation of human epidermal keratinocytes in vitro and in vivo. Recently we found that the N- and C-terminal domains of these proteins show strong sequence homology to loricrin and involucrin, suggesting that SPRR proteins constitute a new class of cornified envelope precursor proteins. Here we show that SPRR proteins are encoded by closely related members of a gene family, consisting of two genes for SPRR1, approximately seven genes for SPRR2, and a single gene for SPRR3. All SPRR genes are closely linked within a 300-kb DNA segment on human chromosome 1 band q21-q22, a region where the related loricrin and involucrin genes have also been mapped. The most characteristic feature of the SPRR gene family resides in the structure of the central segments of the encoded polypeptides that are built up from tandemly repeated units of either eight (SPRR1 and SPRR3) or nine (SPRR2) amino acids with the general consensus *K*PEP**. Sequencing data of the different members, together with their clustered chromosomal organization, strongly suggest that this gene family has evolved from a single progenitor gene by multiple intra- and intergenic duplications. Analysis of the different SPRR subfamilies reveals a gene-specific bias to either intra- or intergenic duplication. We propose that a process of homogenization has acted on the different members of one subfamily, whereas the different subfamilies appear to have diverged from each other, at the levels of both protein structure and gene regulation.     

EGFR regulation of epidermal barrier function

Keratinocyte terminal differentiation is the process that ultimately forms the epidermal barrier that is essential for mammalian survival. This process is controlled, in part, by signal transduction and gene expression mechanisms, and the epidermal growth factor receptor (EGFR) is known to be an important regulator of multiple epidermal functions. Using microarray analysis of a confluent cell density-induced model of keratinocyte differentiation, we identified 2,676 genes that are regulated by epidermal growth factor (EGF), a ligand of the EGFR. We further discovered, and separately confirmed by functional assays, that EGFR activation abrogates all of the known essential processes of keratinocyte differentiation by 1) decreasing the expression of lipid matrix biosynthetic enzymes, 2) regulating numerous genes forming the cornified envelope, and 3) suppressing the expression of tight junction proteins. In organotypic cultures of skin, EGF acted to impair epidermal barrier integrity, as shown by increased transepidermal water loss. As defective epidermal differentiation and disruption of barrier function are primary features of many human skin diseases, we used bioinformatic analyses to identify genes that are known to be associated with skin diseases. Compared with non-EGF-regulated genes, EGF-regulated genes were significantly enriched for skin disease genes. These results provide a systems-level understanding of the actions of EGFR signaling to inhibit keratinocyte differentiation, providing new insight into the role of EGFR imbalance in skin pathogenesis.