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.     

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.     

Epidermal hyperproliferation in mice lacking fatty acid transport protein 4 (FATP4) involves ectopic EGF receptor and STAT3 signaling

Fatty acid transport protein (FATP) 4 is one of a family of six FATPs that facilitate long- and very long-chain fatty acid uptake. Mice lacking FATP4 are born with tight, thick skin and a defective epidermal barrier; they die neonatally due to dehydration and restricted movements. Both the skin phenotype and the lethality are rescued by transgene-driven expression of FATP4 solely in suprabasal keratinocytes. Here we show that Fatp4 mutants exhibit epidermal hyperplasia resulting from an increased number of proliferating suprabasal cells. In addition, barrier formation initiates precociously but never progresses to completion. To investigate possible mechanisms whereby Fatp4 influences skin development, we identified misregulated genes in Fatp4 mutants. Remarkably, three members of the epidermal growth factor (EGF) family (Ereg, Areg, and Epgn) showed increased expression that was associated with elevated epidermal activation of the EGF receptor (EGFR) and STAT3, a downstream effector of EGFR signaling. Both Tyrphostin AG1478, an EGFR tyrosine kinase inhibitor, and curcumin, an inhibitor of both STAT3 and EGFR, attenuated STAT3 activation/nuclear translocation, reduced skin thickening, and partially suppressed the barrier abnormalities. These data identify FATP4 activity as negatively influencing EGFR activation and the resulting STAT3 signaling during normal skin development. These findings have important implications for understanding the pathogenesis of ichthyosis prematurity syndrome, a disease recently shown to be caused by FATP4 mutations.     

Mesenchymal stem cells differentiate into keratinocytes and express epidermal kallikreins: Towards an in vitro model of human epidermis

Epidermal differentiation is a complex process in which keratinocytes go through morphological and biochemical changes in approximately 15 to 30 days. Abnormal keratinocyte differentiation is involved in the pathophysiology of several skin diseases. In this scenario, mesenchymal stem cells (MSCs) emerge as a promising approach to study skin biology in both normal and pathological conditions. Herein, we have studied the differentiation of MSC from umbilical cord into keratinocytes. MSC were cultured in Dulbecco's modified Eagle's medium (DMEM) (proliferation medium) and, after characterization, differentiation was induced by culturing cells in a defined keratinocyte serum-free medium (KSFM) supplemented with epidermal growth factor (EGF) and calcium chloride ions. Cells cultivated in DMEM were used as control. Cultures were evaluated from day 1 to 23, based on the cell morphology, the expression of p63, involucrin and cytokeratins (KRTs) KRT5, KRT10 and KRT14, by quantitative polymerase chain reaction, Western blot analysis or immunofluorescence, and by the detection of epidermal kallikreins activity. In cells grown in keratinocyte serum-free medium with EGF and 1.8 mM calcium, KRT5 and KRT14 expression was shown at the first day, followed by the expression of p63 at the seventh day. KRT10 expression was detected from day seventh while involucrin was observed after this period. Data showed higher kallikrein (KLK) activity in KSFM-cultured cells from day 11th in comparison to control. These data indicate that MSC differentiated into keratinocytes similarly to that occurs in the human epidermis. KLK activity detection appears to be a good methodology for the monitoring the differentiation of MSC into the keratinocyte lineage, providing useful tools for the better understanding of the skin biology.     

Smad7 Modulates Epidermal Growth Factor Receptor Turnover through Sequestration of c-Cbl

Epidermal growth factor (EGF) regulates various cellular events, including proliferation, differentiation, migration, and tumorigenesis. For the maintenance of homeostasis, EGF signaling should be tightly regulated to prevent the aberrant activation. Smad7 has been known as inhibitory Smad that blocks the signal transduction of transforming growth factor β. In the process of cell proliferation or transformation, Smad7 has been shown the opposite activities as a promoter or suppressor depending on cell types or microenvironments. We found that the overexpression of Smad7 in human HaCaT keratinocyte cells and mouse skin tissues elevated EGF receptor (EGFR) activity by impairing ligand-induced ubiquitination and degradation of activated receptor, which is induced by the E3 ubiquitin ligase c-Cbl. The C-terminal MH2 region but not MH1 region of Smad7 is critical for interaction with c-Cbl to inhibit the ubiquitination of EGFR. Interestingly, wild-type Smad7, but not Smad6 or mutant Smad7, destabilized the EGF-induced complex formation of c-Cbl and EGFR. These data suggest a novel role for Smad7 as a promoter for prolonging the EGFR signal in keratinocyte and skin tissue by reducing its ligand-induced ubiquitination and degradation.     

Involvement of connexin43 in the EGF/EGFR signalling during self-renewal and differentiation of neural progenitor cells

Neural progenitor cells (NPCs) are sensitive to epidermal growth factor (EGF), which is essential for their self-renewal. Recently we showed that high level of connexin43 (Cx43) expression and gap junctional intercellular communication (GJIC) are also required to maintain NPCs in a proliferative state. In this study the connection between EGF/EGFR signalling and Cx43 expression was investigated during proliferation and differentiation of cultured ReNcell VM197 human NPCs. We found that EGF, but not basic fibroblast growth factor (bFGF), strongly stimulated both Cx43 expression and GJIC in proliferating cells. This stimulatory effect was blocked by AG1478, a specific inhibitor for EGFR kinase. Notably, knockdown of Cx43 strongly inhibited the cell proliferation promoted by EGF/EGFR signalling. High sensitivity to EGF was still maintained in differentiated NPCs. Administration of EGF to differentiating cells led to a pronounced increase (9-fold) of Cx43 expression and a re-induction of proliferation. This strong impact of EGF was found to correlate with a surprisingly massive 60-fold up-regulation of EGFR expression in differentiated cells. Our data argue for a mutual regulation between Cx43 expression and EGF/EGFR signalling during self-renewal and differentiation of NPCs.     

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.     

HaCaT keratinocyte migration is dependent on epidermal growth factor receptor signaling and glycogen synthase kinase-3alpha

After epithelial disruption by tissue injury, keratinocytes migrate from the wound edge into a provisional matrix. This process is stimulated by growth factors that signal through epidermal growth factor (EGF) receptor, including EGF, heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor-alpha (TGF-alpha), and by for example keratinocyte growth factor (KGF) and TGF-beta1 that function through different receptors. We have previously shown that keratinocyte migration induced by EGF or staurosporine is dependent on the activity of glycogen synthase kinase-3 (GSK-3). In the present study, we show that keratinocyte migration induced by TGF-beta1, KGF, EGF, TGF-alpha and staurosporine depends on EGFR signaling, involves autocrine HB-EGF expression and is potently blocked by GSK-3 inhibitors SB-415286 and LiCl. Inhibition of GSK-3 also retards wound reepithelialization in vivo in mice. Moreover, inhibition of GSK-3 activity prevented cell rounding that is an early event in EGFR-mediated keratinocyte migration. Isoform-specific GSK-3alpha and GSK-3beta knockdown and overexpression experiments with siRNAs and adenoviral constructs, respectively, revealed that GSK-3alpha is required for keratinocyte migration, whereas excessive activity of GSK-3beta is inhibitory. Thus, induction of keratinocyte migration is conveyed through EGFR, promoted by endogenous HB-EGF and requires GSK-3alpha activity.     

A novel function of angiotensin II in skin wound healing. Induction of fibroblast and keratinocyte migration by angiotensin II via heparin-binding epidermal growth factor (EGF)-like growth factor-mediated EGF receptor transactivation

The role of angiotensin II (Ang II) in the control of systemic blood pressure and volume homeostasis is well known and has been extensively studied. Recently, Ang II was suggested to also have a function in skin wound healing. In the present study, the in vivo function of Ang II in skin wound healing was investigated using Ang II type 1 receptor (AT1R) knock-out mice. Wound healing in these mice was found to be markedly delayed. Keratinocytes and fibroblasts play important roles in wound healing, and thus the effect of Ang II on the migration of these cells was examined. Ang II stimulated keratinocyte and fibroblast migration in a dose-dependent manner. It has been reported that G protein-coupled receptor (GPCR) activation induces epidermal growth factor (EGF) receptor (EGFR) transactivation through the shedding of heparin-binding EGF-like growth factor (HB-EGF). As AT1R is a GPCR, it was hypothesized that Ang II-induced keratinocyte and fibroblast migration is mediated by EGFR transactivation. Ang II induced EGFR phosphorylation, which was inhibited by an AT1R antagonist, HB-EGF neutralizing antibody, and an HB-EGF antagonist in both keratinocytes and in fibroblasts. Moreover, Ang II-induced migration of keratinocytes and fibroblasts was also prevented by these inhibitors. Taken together, these findings clearly demonstrate, for the first time, that Ang II plays an important role in skin wound healing and that it functions by accelerating keratinocyte and fibroblast migration in a process mediated by HB-EGF shedding.     

Expression of a dominant negative mutant of epidermal growth factor receptor in the epidermis of transgenic mice elicits striking alterations in hair follicle development and skin structure.

Epidermal growth factor receptor (EGFR) is a key regulator of keratinocyte biology. However, the physiological role of EGFR in vivo has not been well established. To analyze the role of EGFR in skin, we have generated transgenic mice expressing an EGFR dominant negative mutant in the basal layer of epidermis and outer root sheath of hair follicles. Mice expressing the mutant receptor display short and waved pelage hair and curly whiskers during the first weeks of age, but subsequently pelage and vibrissa hairs become progressively sparser and atrophic. Eventually, most mice present severe alopecia. Histological examination of the skin of transgenic mice shows striking alterations in the development of hair follicles, which fail to enter into catagen stage. These alterations eventually lead to necrosis and disappearance of the follicles, accompanied by strong infiltration of the skin with inflammatory elements. The interfollicular epidermis of these mice shows marked hyperplasia, expression of hyperproliferation-associated keratin K6 and increased 5-bromo-2-deoxyuridine incorporation. EGFR function was inhibited in transgenic skin keratinocytes, since in vivo and in vitro autophosphorylation of EGFR was almost completely abolished on EGF stimulation. These results implicate EGFR in the control of hair cycle progression, and provide new information about its role in epidermal growth and differentiation.     

Expression and Functional Role of Sox9 in Human Epidermal Keratinocytes

In this study, we investigated the expression and putative role of Sox9 in epidermal keratinocyte. Immunohistochemical staining showed that Sox9 is predominantly expressed in the basal layer of normal human skin epidermis, and highly expressed in several skin diseases including psoriasis, basal cell carcinoma, keratoacanthoma and squamous cell carcinoma. In calcium-induced keratinocyte differentiation model, the expression of Sox9 was decreased in a time dependent manner. When Sox9 was overexpressed using a recombinant adenovirus, cell growth was enhanced, while the expression of differentiation-related genes such as loricrin and involucrin was markedly decreased. Similarly, when rat skin was intradermally injected with the adenovirus expressing Sox9, the epidermis was thickened with increase of PCNA positive cells, while the epidermal differentiation was decreased. Finally, UVB irradiation induced Sox9 expression in cultured human epidermal keratinocytes, and keratinocytes are protected from UVB-induced apoptosis by Sox9 overexpression. Together, these results suggest that Sox9 is an important regulator of epidermal keratinocytes with putative pro-proliferation and/or pro-survival functions, and may be related to several cutaneous diseases that are characterized by abnormal differentiation and hyperproliferation.     

RhoB protects human keratinocytes from UVB-induced apoptosis through epidermal growth factor receptor signaling

Exposure of the skin to UVB light results in the formation of DNA photolesions that can give rise to cell death, mutations, and the onset of carcinogenic events. Specific proteins are activated by UVB and then trigger signal transduction pathways that lead to cellular responses. An alteration of these signaling molecules is thought to be a fundamental event in tumor promotion by UVB irradiation. RhoB, encoding a small GTPase has been identified as a DNA damage-inducible gene. RhoB is involved in epidermal growth factor (EGF) receptor trafficking, cytoskeletal organization, cell transformation, and survival. We have analyzed the regulation of RhoB and elucidated its role in the cellular response of HaCaT keratinocytes to relevant environmental UVB irradiation. We report here that the activated GTP-bound form of RhoB is increased rapidly within 5 min of exposure to UVB, and then RhoB protein levels increased concomitantly with EGF receptor (EGFR) activation. Inhibition of UVB-induced EGFR activation prevents RhoB protein expression and AKT phosphorylation but not the early activation of RhoB. Blocking UVB-induced RhoB expression with specific small interfering RNAs inhibits AKT and glycogen synthase kinase-3beta phosphorylation through inhibition of EGFR expression. Moreover, down-regulation of RhoB potentiates UVB-induced cell apoptosis. In contrast, RhoB overexpression protects keratinocytes against UVB-induced apoptosis. These results indicated that RhoB is regulated upon UVB exposure by a two-step process consisting of an early EGFR-independent RhoB activation followed by an EGFR-dependent induction of RhoB expression. Moreover, we have demonstrated that RhoB is essential in regulating keratinocyte cell survival after UVB exposure, suggesting its potential role in photocarcinogenesis.     

EGFR-driven up-regulation of decoy receptor 3 in keratinocytes contributes to the pathogenesis of psoriasis

Decoy receptor 3 (DcR3) is a soluble receptor of Fas ligand (FasL), LIGHT (TNFSF14) and TNF-like molecule 1A (TL1A) and plays pleiotropic roles in many inflammatory and autoimmune disorders and malignant diseases. In cutaneous biology, DcR3 is expressed in primary human epidermal keratinocytes and is upregulated in skin lesions in psoriasis, which is characterized by chronic inflammation and angiogenesis. However, the regulatory mechanisms of DcR3 over-expression in skin lesions of psoriasis are unknown. Here, we demonstrate that DcR3 can be detected in both dermal blood vessels and epidermal layers of psoriatic skin lesions. Analysis of serum samples showed that DcR3 was elevated, but FasL was downregulated in psoriatic patients compared with normal individuals. Additional cell studies revealed a central role of epidermal growth factor receptor (EGFR) in controlling the basal expression of DcR3 in keratinocytes. Activation of EGFR by epidermal growth factor (EGF) and transforming growth factor (TGF)-α strikingly upregulated DcR3 production. TNF-α?enhanced DcR3 expression in both keratinocytes and endothelial cells compared with various inflammatory cytokines involved in psoriasis. Additionally, TNF-α-enhanced DcR3 expression in keratinocytes was inhibited when EGFR was knocked down or EGFR inhibitor was used. The NF-κB pathway was critically involved in the molecular mechanisms underlying the action of EGFR and inflammatory cytokines. Collectively, the novel regulatory mechanisms of DcR3 expression in psoriasis, particularly in keratinocytes and endothelial cells, provides new insight into the pathogenesis of psoriasis and may also contribute to the understanding of other diseases that involve DcR3 overexpression.