Increased expression of keratin 16 causes anomalies in cytoarchitecture and keratinization in transgenic mouse skin

Injury to epidermis and other stratified epithelia triggers profound but transient changes in the pattern of keratin expression. In postmitotic cells located at the wound edge, a strong induction of K6, K16, and K17 synthesis occurs at the expense of the keratins produced under the normal situation. The functional significance of these alterations in keratin expression is not known. Here, we report that overexpression of a wild-type human K16 gene in a tissue-specific fashion in transgenic mice causes aberrant keratinization of the hair follicle outer root sheath and proximal epidermis, and it leads to hyperproliferation and increased thickness of the living layers (acanthosis), as well as cornified layers (hyperkeratosis). The pathogenesis of lesions in transgenic mouse skin begins with a reorganization of keratin filaments in postmitotic keratinocytes, and it progresses in a transgene level-dependent fashion to include disruption of keratinocyte cytoarchitecture and structural alterations in desmosomes at the cell surface. No evidence of cell lysis could be found at the ultrastructural level. These results demonstrate that the disruption of the normal keratin profile caused by increased K16 expression interferes with the program of terminal differentiation in outer root sheath and epidermis. They further suggest that when present at sufficiently high intracellular levels, K16, along with K6 and K17, appear capable of inducing a reorganization of keratin filaments in the cytoplasm of skin epithelial cells.     

The relationships among adhesion, stratification and differentiation in keratinocytes

Epidermis is a self-renewing, multilayered tissue composed primarily of keratinocytes. The epidermal keratinocyte follows a terminal differentiation pathway that under normal circumstances is tightly linked to its position within the epidermis and culminates in the formation of the protective barrier (stratum corneum) that constitutes the outermost layer of skin. Strong but pliant adhesive mechanisms are essential for normal functioning of the epidermis. In the epidermis, adhesion is mediated primarily by four structures: hemidesmosomes and focal adhesions, which function in cell-matrix adhesion, and desmosomes and adherens junctions, which function in cell-cell adhesion. In this review we concentrate on the transmembrane components of these structures, which are thought to mediate directly the adhesive function. Members of the integrin family of adhesion molecules comprise the transmembrane components of hemidesmosomes and focal adhesions, although hemidesmosomes also have a second, unrelated transmembrane molecule known as 'bullous pemphigoid antigen 2'. Members of the cadherin family are the transmembrane constituents of desmosomes and adherens junctions. Desmosomes consistently contain two types of cadherins (desmoglein and desmocollin), while adherens junctions may contain only one type of cadherin (E- or P-cadherin). Expression of most of the transmembrane components varies with the position of the keratinocyte within the epidermis and thus may reflect the degree of epidermal differentiation. All of the integrin subunits have been localized predominantly to the basal layer. In contrast, the cadherins show very complex expression patterns throughout the epidermis. Desmogleins and desmocollins (the desmosomal cadherins) are each encoded by three genes, and the expression of each gene is limited to certain epidermal layers. With respect to the cadherins of the adherens junction, it has been shown that E-cadherin is present throughout the epidermis, while P-cadherin is limited to the basal layer. Interestingly, these complex expression patterns of integrins and cadherins within the epidermis may not simply be passive events in differentiation; rather, evidence is accumulating that adhesion molecules can exert a dynamic role in epidermal differentiation/stratification. For example, decreased adhesion to extracellular matrix, induced by changes in one or more integrins, appears to be a signal that induces certain differentiation-related events. Even more profound effects on epidermal morphogenesis have been demonstrated for the cadherins. E- and/or P-cadherin is required not only to initiate normal intercellular junction formation but also for the subsequent development of a stratified epithelium. Thus, the findings to date with both integrins and cadherins suggest that adhesion molecules may function not just as direct mediators of adhesion, but also as regulators of epidermal stratification, differentiation, and morphogenesis.     

Regulation of C-X-C chemokine gene expression by keratin 17 and hnRNP K in skin tumor keratinocytes

High levels of the intermediate filament keratin 17 (K17) correlate with a poor prognosis for several types of epithelial tumors. However, the causal relationship and underlying mechanisms remain undefined. A recent study suggested that K17 promotes skin tumorigenesis by fostering a specific type of inflammation. We report here that K17 interacts with the RNA-binding protein hnRNP K, which has also been implicated in cancer. K17 is required for the cytoplasmic localization of hnRNP K and for its role in regulating the expression of multiple pro-inflammatory mRNAs. Among these are the CXCR3 ligands CXCL9, CXCL10, and CXCL11, which together form a signaling axis with an established role in tumorigenesis. The K17–hnRNP K partnership is regulated by the ser/thr kinase RSK and required for CXCR3-dependent tumor cell growth and invasion. These findings functionally integrate K17, hnRNP K, and gene expression along with RSK and CXCR3 signaling in a keratinocyte-autonomous axis and provide a potential basis for their implication in tumorigenesis.     

IL-22 inhibits epidermal differentiation and induces proinflammatory gene expression and migration of human keratinocytes

IL-22 belongs to a family of cytokines structurally related to IL-10, including IL-19, IL-20, IL-24, and IL-26. In contrast to IL-10, IL-22 has proinflammatory activities. IL-22 signals through a class II cytokine receptor composed of an IL-22-binding chain, IL-22RA1, and the IL-10RB subunit, which is shared with the IL-10R. In the present study, we show that short-term cultured human epidermal keratinocytes express a functional IL-22R but no IL-10R. Accordingly, IL-22 but not IL-10 induces STAT3 activation in keratinocytes. Using a cDNA array screening approach, real-time RT-PCR, and Western blot analysis, we demonstrate that IL-22 up-regulates, in a dose-dependent manner, the expression of S100A7, S100A8, S100A9, a group of proinflammatory molecules belonging to the S100 family of calcium-binding proteins, as well as the matrix metalloproteinase 3, the platelet-derived growth factor A, and the CXCL5 chemokine. In addition, IL-22 induces keratinocyte migration in an in vitro injury model and down-regulates the expression of at least seven genes associated with keratinocyte differentiation. Finally, we show that IL-22 strongly induces hyperplasia of reconstituted human epidermis. Taken together, these results suggest that IL-22 plays an important role in skin inflammatory processes and wound healing.     

Chronic inflammatory disease alters adhesion molecule requirements for acute neutrophil emigration in mouse skin

Mutant mice triply deficient in ICAM-1, E-selectin, and P-selectin did not develop the neutrophilic skin lesions that spontaneously arise in mutants doubly deficient in E-selectin and P-selectin. Thus, ICAM-1 is essential to skin disease resulting from endothelial selectin deficiency. During experimental dermatitis, acute neutrophil emigration was completely prevented in young mice deficient in both selectins (E/P and E/P/I mutants). However, older E/P mutants with spontaneous skin lesions displayed an endothelial selectin-independent pathway for acute neutrophil emigration. In contrast, emigration remained compromised in E/P/I mutants and CD18 mutants regardless of age or lesions. Experimentally induced chronic lesions elicited this pathway for acute emigration in young E/P mutants. Thus, an endothelial selectin-independent pathway for acute neutrophil emigration is induced in E/P mice by chronic inflammation at distant sites, and this pathway may contribute to skin disease resulting from endothelial selectin deficiency.     

Onset of re-epithelialization after skin injury correlates with a reorganization of keratin filaments in wound edge keratinocytes: defining a potential role for keratin 16

Injury to stratified epithelia causes a strong induction of keratins 6 (K6) and 16 (K16) in post-mitotic keratinocytes located at the wound edge. We show that induction of K6 and K16 occurs within 6 h after injury to human epidermis. Their subsequent accumulation in keratinocytes correlates with the profound reorganization of keratin filaments from a pan-cytoplasmic distribution to one in which filaments are aggregated in a juxtanuclear location, opposite to the direction of cell migration. This filament reorganization coincides with additional cytoarchitectural changes and the onset of re-epithelialization after 18 h post-injury. By following the assembly of K6 and K16 in vitro and in cultured cells, we find that relative to K5 and K14, a well- characterized keratin pair that is constitutively expressed in epidermis, K6 and K16 polymerize into short 10-nm filaments that accumulate near the nucleus, a property arising from K16. Forced expression of human K16 in skin keratinocytes of transgenic mice causes a retraction of keratin filaments from the cell periphery, often in a polarized fashion. These results imply that K16 may not have a primary structural function akin to epidermal keratins. Rather, they suggest that in the context of epidermal wound healing, the function of K16 could be to promote a reorganization of the cytoplasmic array of keratin filaments, an event that precedes the onset of keratinocyte migration into the wound site.     

RhoB affects macrophage adhesion, integrin expression and migration

Rho GTPases regulate multiple cellular responses, including cell motility and cell cycle progression. The Rho isoform RhoB represses transformation and affects endosomal trafficking, but its effects on cell adhesion and migration have not been investigated in detail. Here we show that RhoB-null macrophages are more rounded than wild-type macrophages on fibronectin and uncoated glass, and have reduced adhesion to ICAM-1 and glass but not fibronectin. This correlated with lower cell surface expression of beta2 and beta3 integrins but not beta1 integrin. RhoB-null cells migrated faster than Wt cells on fibronectin, consistent with their smaller spread area, but slower than Wt cells on glass, reflecting their reduced adhesion. C3 transferase, which inhibits RhoA, RhoB and RhoC, induced cell spreading but this effect was reduced in RhoB-null cells. However, RhoB is not required for assembly of podosomes, which are integrin-based adhesion sites, whereas C3 transferase induced a decrease in podosomes and defects in tail retraction. Since macrophages do not express RhoC, these effects of C3 transferase are due to inhibition of RhoA rather than RhoB. Our results suggest that RhoB affects cell shape and migration by regulating surface integrin levels.     

Ammonium induces aberrant blastocyst differentiation,metabolism, pH regulation,gene expression and subsequently alters fetal development in the mouse

The presence of ammonium in the culture medium has significant detrimental effects on the regulation of embryo physiology and genetics. Ammonium levels build up linearly over time in the culture medium when media containing amino acids are incubated at 37 degrees C. Ammonium in the culture media significantly reduces blastocyst cell number, decreases inner cell mass development, increases apoptosis, perturbs metabolism, impairs the ability of embryos to regulate intracellular pH, and alters the expression of the imprinted gene H19. In contrast, the rate of blastocyst development and blastocyst morphology appear to be normal. The transfer of blastocysts exposed to ammonium results in a significant reduction in the ability to establish a pregnancy. Furthermore, of those embryos that manage to implant, fetal growth is significantly impaired. Embryos exposed to 300 microM ammonium are retarded by 1.5 days developmentally at Day 15 of pregnancy. It is therefore essential that culture conditions for mammalian embryos are designed to minimize the buildup of ammonium to prevent abnormalities in embryo physiology, genetic regulation, pregnancy, and fetal development.     

Polymerized laminin-332 matrix supports rapid and tight adhesion of keratinocytes, suppressing cell migration

Laminin-332 (α3?3γ2) (Lm332) supports the stable anchoring of basal keratinocytes to the epidermal basement membrane, while it functions as a motility factor for wound healing and cancer invasion. To understand these contrasting activities of Lm332, we investigated Lm332 matrices deposited by normal human keratinocytes and other Lm332-expressing cell lines. All types of the cells efficiently deposited Lm332 on the culture plates in specific patterns. On the contrary, laminins containing laminin ?1 and/or γ1 chains, such as Lm511 and Lm311, were not deposited on the culture plates even if secreted into culture medium. The Lm332 deposition was not inhibited by function-blocking antibodies to the α3 and α6 integrins but was inhibited by sodium selenate, suggesting that sulfated glycosaminoglycans on cell surface, e.g. heparan sulfate proteoglycans, might be involved in the process. HEK293 cells overexpressing exogenous Lm332 (Lm332-HEK) almost exclusively deposited Lm332 on the plates. The deposited Lm332 matrix showed a mesh-like network structure as analyzed by electron microscopy, suggesting that Lm332 was highly polymerized. When biological activity was analyzed, the Lm332 matrix rather suppressed the migration of keratinocytes as compared with purified Lm332, which highly promoted the cell migration. The Lm332 matrix supported adhesion of keratinocytes much more strongly and stably than purified Lm332. Integrin α3?1 bound to the Lm332 matrix at a three times higher level than purified Lm332. Normal keratinocytes prominently showed integrin α6?4-containing, hemidesmosome-like structures on the Lm332 matrix but not on the purified one. These results indicate that the polymerized Lm332 matrix supports stable cell adhesion by interacting with both integrin α6?4 and α3?1, whereas unassembled soluble Lm332 supports cell migration.     

Forced expression of HOXA13 confers oncogenic hallmarks to esophageal keratinocytes

HOXA13 overexpression has been detected in human ESCC tissue and high HOXA13 protein expression is correlated with a shorter median survival time in ESCC patients. Although aberrant expression of HOXA13 in ESCC has thus been established, little is known regarding the functional consequences thereof. The present study aimed to examine to what extent aberrant HOXA13 might drive carcinogenesis in esophageal keratinocytes. To this end, we overexpressed HOXA13 in a non-transformed human esophageal cell line EPC2-hTERT, performed gene expression profiling to identify key processes and functions, and performed functional experiments. We found that HOXA13 expression confers oncogenic hallmarks to esophageal keratinocytes. It provides proliferation advantage to keratinocytes, reduces sensitivity to chemical agents, regulates MHC class I expression and differentiation status and promotes cellular migration. Our data indicate a crucial role of HOXA13 at early stages of esophageal carcinogenesis.     

Aluminum fluoride stimulates inositol phosphate metabolism and inhibits expression of differentiation markers in mouse keratinocytes.

Mouse keratinocytes are induced to differentiate in vitro by elevating the level of extracellular calcium from 0.05 mM, where keratinocytes express a basal cell phenotype, to greater than 0.10 mM, where they express the differentiated phenotype. This process has been associated with a rapid, sustained increase in inositol phosphate (InsP) turnover, which precedes the expression of differentiation-specific proteins. In 0.05 mM Ca2+ medium, aluminum and fluoride salts (AIF4-), which combine to activate nonspecifically heterotrimeric guanine nucleotide-binding (G) proteins, cause a concentration-dependent increase in InsP metabolism in keratinocytes, and generate elevated intracellular diacylglycerol levels. This is associated with an inhibition of cell growth. Treatment with both AIF4- and Ca2+ greater than 0.10 mM resulted in an additive increase in InsP turnover, implying the presence of at least two responsive InsP pools. AIF4- inhibited the expression of differentiation markers induced by Ca2+ greater than 0.10 mM and altered the morphology of keratinocytes from squamous to dendritic, which was reversible upon withdrawal of AIF4-. Neoplastic keratinocytes, in which basal levels of InsP metabolism are higher than in normal cells, do not differentiate in response to Ca2+. Neoplastic keratinocytes responded to AIF-4 treatment with an even greater rise in InsP metabolism. AIF-4 also inhibited cell growth and reversibly altered morphology in neoplastic keratinocytes. These data suggest that InsP metabolism in keratinocytes is at least partially regulated by a G protein mechanism. Furthermore, an increase in InsP metabolism is not sufficient to stimulate differentiation and may be inhibitory to differentiation if exceeding limited increases. However, these observations cannot exclude the possibility that other AIF-4 stimulated pathways involving G or non-G proteins can also influence keratinocyte biology.     

A human epidermal differentiation-specific keratin gene is regulated by calcium but not negative modulators of differentiation in transgenic mouse keratinocytes.

Keratins K1 and K10 represent the major differentiation products of the maturing epidermal keratinocytes. Primary epidermal cell cultures from newborn K1 transgenic mice containing a 12-kilobase human K1 genomic fragment were established in order to examine the expression of both human and mouse K1 in the presence of known modulators of epidermal differentiation. Elevated levels of Ca2+ in the culture medium induced both mouse K1 and human K1. Supplementing the medium with retinoic acid or 12-O-tetradecanoylphorbol-13-acetate or introducing a Harvey viral ras oncogene (v-rasHa) into the cells completely suppressed mouse K1 but not human K1. Our results suggest that: (a) the human 12-kilobase insert contains all the necessary cis-acting elements to respond to the Ca2+ signal, and (b) other cis-acting elements, not present within this insert, may function independently to regulate the response of K1 to retinoids, 12-O-tetradecanoylphorbol-13-acetate, and v-rasHa transformation. This transgenic model provides an approach to identify elements required for the regulation of an epidermal differentiation-specific gene.     

Differential expression of keratin genes during mouse development

Suprabasal layers of the newborn mouse epidermis contain two mRNAs of 2.0 and 2.4 kb which are translated into keratins of 59 and 67 kDa, respectively. To study their expression during development, cDNA sequences corresponding to the 2.0- and the 2.4-kb mRNAs were cloned, characterized by hybridization selection assay, and used as probes to detect keratin sequences in polyadenylated RNA from Day 11, 13, 15, and 17 embryos. In RNA from Day 11 of gestation, two RNAs of 2.8 and 1.8 kb were identified. They were found to have homologies with both epidermal RNAs, suggesting that they are coding for proteins of the keratin family. These two sequences were not detected in sample of later stages. RNAs comigrating with the two epidermal keratin RNAs were identified only in Day 15 and 17 embryos indicating that their expression was induced between Day 13 and 15. Finally, the localization of the 59-kDa keratin mRNA was examined by in situ hybridization. The spinous and granulous cell layers were found to be heavily covered with grains while other regions of the tissue sections were unlabeled. All these results support the hypothesis of a sequential expression of keratins during differentiation of epidermal cells and suggest that proteins related to the keratins expressed specifically in keratinizing cells are expressed earlier during development.     

Effects of prostaglandins on the spreading, adhesion and migration of mouse peritoneal macrophages

The effects of prostaglandins on the properties of mouse peritoneal macrophages namely spreading, adhesion and migration were investigated. PGE₁ and PGE₂ inhibit the spreading and adhesion of complete Freund's Adjuvant induced peritoneal macrophages significantly at concentrations of 1 ng per ml and above whereas they enhance the migration of these cells at concentrations of 100 ng per ml and above. PGA₂ and PGB₂ are less potent as they inhibit spreading and adhesion only at a concentration of 1 μg per ml. At this concentration PGB₂ enhances migration whereas PGA₂ has no effect. PGF_2α has no effect on the spreading, adhesion and migration of macrophages in the concentration range of 0.1 ng to 1,000 ng per ml.     

Coordinate control by vitamin A of keratin gene expression in human keratinocytes

In the present study, we examine the effects of vitamin A on keratin protein and mRNA levels in human keratinocytes. In epidermal keratinocytes, the levels of keratins 5, 6, 14, and 17 decrease and keratins 13 and 19 increase in response to increasing concentrations of a potent synthetic trans-retinoic acid analog, arotinoid Ro 13-6298. In tracheal keratinocytes, a similar suppression is observed for keratins 5, 6, 14, 17, and 18 and an increase in keratin 19. Both induction and suppression responses show identical kinetics and both processes are half-maximal at 5 nM arotinoid and maximal at 10 nM. Utilizing cDNAs specific for keratins 5, 6, 13, and 19, we demonstrate that the mRNA levels for these keratins change coordinately with the corresponding amount of keratin protein, indicating that the control of keratin protein expression most likely resides at the level of mRNA synthesis and/or degradation. The identical kinetics for all of the responses, both inductive and suppressive, suggests that a common mechanism controls the expression of these genes. These results indicate that vitamin A produces more sweeping changes in keratinocyte function than previously appreciated in that many and perhaps all keratins are modulated by vitamin A. Moreover, these responses are 10- to 100-fold less sensitive to retinoid than the process of envelope formation, suggesting that at least two sets of processes with different sensitivities to vitamin A are present in keratinocytes.     

Changes in keratin gene expression during terminal differentiation of the keratinocyte

Cells of the inner layers of the epidermis contain small keratins (46-58K), whereas the cells of the outer layers contain large keratins (63-67K) in addition to small ones. The changes in keratin composition that take place within each cell during the course of its terminal differentiation result largely from changes in synthesis. Cultured epidermal cells resemble cells of the inner layers of the epidermis in synthesizing only small keratins. The cultured cells possess translatable mRNA only for small keratins, whereas mRNA extracted from whole epidermis can be translated into both large and small keratins. As no synthesis takes place in the outermost layer of the epidermis (stratum corneum), the keratins of this layer must be synthesized earlier, but in some cases they then become smaller: this presumably occurs by post-translational processing of the molecules during the final stages of differentiation. Stratified squamous epithelia of internal organs do not form a typical stratum corneum and do not make the large keratins characteristic of epidermis. Their keratins are also different from those of cultured keratinocytes, implying that they have embarked on an alternate route of terminal keratin synthesis.     

A role for disulfide bonding in keratin intermediate filament organization and dynamics in skin keratinocytes

We recently reported that a trans-dimer, homotypic disulfide bond involving Cys367 in keratin 14 (K14) occurs in an atomic-resolution structure of the interacting K5/K14 2B domains and in keratinocyte cell lines. Here we show that a sizable fraction of the K14 and K5 protein pools participates in interkeratin disulfide bonding in primary cultures of mouse skin keratinocytes. By comparing the properties of wild-type K14 with a completely cysteine-free variant thereof, we found that K14-dependent disulfide bonding limited filament elongation during polymerization in vitro but was necessary for the genesis of a perinuclear-concentrated network of keratin filaments, normal keratin cycling, and the sessile behavior of the nucleus and whole cell in keratinocytes studied by live imaging. Many of these phenotypes were rescued when analyzing a K14 variant harboring a single Cys residue at position 367. These findings establish disulfide bonding as a novel and important mechanism regulating the assembly, intracellular organization, and dynamics of K14-containing intermediate filaments in skin keratinocytes.