Analysis of heterogeneity of human keratinocyte populations by their adhesion to substrate and the keratin 19 to actin ratio

Keratin 19 is reported to be a marker for skin stem cells and that assumes an independent subpopulation of these cells in keratinocyte population. In addition, keratinocytes have natural affinity to extracellular matrix proteins. The aim of this work was to reveal subpopulations ofkeratinocytes cultivated on the substrates of collagen I type, laminin-2/4 and fibronectin, and distinguished by keratin 19/actin ratio (G/R). The area of cell projection on a substrate, perimeter, the spreading coefficient and G/R were measured. Both on fibronectin and laminin-2/4, keratinocyte populations were morphologically homogeneous with large cells comprising 12 and 20 percents of the population, respectively. On fibronectin, correlation between morphological parameters of the cells and the keratin 19/actin ratio was not found. The cells growth on collagen behaved as a heterogeneous population, with the large cells compressing more than 50 percents of the population. An average, the size of the cells growing on collagen was twice as large as than of the cells cultivated on fibronectin. On the other hand, correlation between the cell size and G/R was revealed in the cells growth both on collagen and laminin-2/4. The cells with lower G/R value display a larger size and extent of spreading. We assume that this correlation may be determined by alphal and alpha2, integrins characteristic of these cells. Our results cast doubts on whether stem cells are present in the culture of human skin keratinocytes.     

Laminin-5 Inhibits Human Keratinocyte Migration

Laminin-5 (previously known as kalinin, epiligrin, and nicein) is an adhesive protein localized to the anchoring filaments within the lamina lucida space of the basement membrane zone lying between the epidermis and dermis of human skin. Anchoring filaments are structures within the lamina lucida and lie immediately beneath the hemidesmosomes of the overlying basal keratinocytes apposed to the basement membrane zone. Human keratinocytes synthesize and deposit laminin-5. Laminin-5 is present at the wound edge during reepithelialization. In this study, we demonstrate that laminin-5, a powerful matrix attachment factor for keratinocytes, inhibits human keratinocyte migration. We found that the inhibitory effect of laminin-5 on keratinocyte motility can be reversed by blocking the α3 integrin receptor. Laminin-5 inhibits keratinocyte motility driven by a collagen matrix in a concentration-dependent fashion. Using antisense oligonucleotides to the α3 chain of laminin-5 and an antibody that inhibits the cell binding function of secreted laminin-5, we demonstrated that the endogenous laminin-5 secreted by the keratinocyte also inhibits the keratinocyte's own migration on matrix. These findings explain the hypermotility that characterizes keratinocytes from patients who have forms of junctional epidermolysis bullosa associated with defects in one of the genes encoding for laminin-5 chains, resulting in low expression and/or functional inadequacy of laminin-5 in these patients. These studies also suggest that during reepithelialization of human skin wounds, the secreted laminin-5 stabilizes the migrating keratinocyte to establish the new basement membrane zone.     

Differential regulation of alpha6beta4 integrin by PKC isoforms in murine skin keratinocytes

In mammalian epidermis, alpha6beta4 integrin is expressed exclusively on the basal layer localized to the hemidesmosomes, where it interacts extracellularly with the laminin-5 ligand. During differentiation, loss of alpha6beta4 is associated with keratinocyte detachment from the basement membrane and upward migration. The protein kinase C (PKC) family of isoforms participates in regulation of integrin function and is linked to skin differentiation. Exposure of primary murine keratinocytes to PKC activators specifically downregulates alpha6beta4 expression. Utilizing recombinant adenoviruses, we selectively overexpressed skin PKC isoforms in primary keratinocytes. PKCdelta and PKCzeta induced downregulation of alpha6beta4 protein expression, leading to reduced keratinocyte attachment to laminin-5 and enhanced gradual detachment from the underlying matrix. In contrast, PKCalpha upregulated alpha6beta4 protein expression, leading to increased keratinocyte attachment to laminin-5 and to the underlying matrix. Altogether, these results suggest distinct roles for specific PKC isoforms in alpha6beta4 functional regulation during the early stages of skin differentiation.     

Targeted deletion of the integrin beta4 signaling domain suppresses laminin-5-dependent nuclear entry of mitogen-activated protein kinases and NF-kappaB, causing defects in epidermal growth and migration

The alpha6beta4 integrin-a laminin-5 receptor-mediates assembly of hemidesmosomes and recruitment of Shc and phosphoinositide 3-kinase through the unique cytoplasmic extension of beta4. Mice carrying a targeted deletion of the signaling domain of beta4 develop normally and do not display signs of skin fragility. The epidermis of these mice contains well-structured hemidesmosomes and adheres stably to the basement membrane. However, it is hypoplastic due to reduced proliferation of basal keratinocytes and undergoes wound repair at a reduced rate. Keratinocytes from beta4 mutant mice undergo extensive spreading but fail to proliferate and migrate in response to epidermal growth factor (EGF) on laminin-5. EGF causes significant phosphorylation of extracellular signal-regulated kinase (ERK) and Jun N-terminal protein kinase (JNK) and phosphorylation and degradation of IkappaB in beta4 mutant cells adhering to laminin-5. Unexpectedly, however, ERK, JNK, and NF-kappaB remain in the cytoplasm in beta4 mutant cells on laminin-5, whereas they enter effectively into the nucleus in the same cells on fibronectin or in wild-type cells on both matrix proteins. Inhibitor studies indicate that alpha6beta4 promotes keratinocyte proliferation and migration through its effect on NF-kappaB and P-JNK. These findings provide evidence that beta4 signaling promotes epidermal growth and wound healing through a previously unrecognized effect on nuclear translocation of NF-kappaB and mitogen-activated protein kinases.     

The role of alpha3beta1 integrin in determining the supramolecular organization of laminin-5 in the extracellular matrix of keratinocytes

Analyses of mice with targeted deletions in the genes for alpha3 and beta1 integrin suggest that the alpha3beta1 integrin heterodimer likely determines the organization of the extracellular matrix within the basement membrane of skin. Here we tested this hypothesis using keratinocytes derived from alpha3 integrin-null mice. We have compared the organizational state of laminin-5, a ligand of alpha3beta1 integrin, in the matrix of wild-type keratinocytes with that of laminin-5 in the matrix of alpha3 integrin-null cells. Laminin-5 distributes diffusely in arc structures in the matrix of wild-type mouse keratinocytes, whereas laminin-5 is organized into linear, spike-like arrays by the alpha3 integrin-null cells. The fact that alpha3 integrin-null cells are deficient in their ability to assemble a proper laminin-5 matrix is also shown by their failure to remodel laminin-5 when plated onto surfaces coated with purified laminin-5 protein. In sharp contrast, wild-type keratinocytes organize exogenously added laminin-5 into discrete ring-like organizations. These findings led us next to assess whether differences in laminin-5 organization in the matrix of the wild-type and alpha3 integrin-null cells impact cell behavior. Our results indicate that alpha3 integrin-null cells are more motile than their wild-type counterparts and leave extensive trails of laminin-5 over the surface on which they move. Moreover, HEK 293 cells migrate significantly more on the laminin-5-rich matrix derived from the alpha3 integrin-null cells than on the wild-type keratinocyte laminin-5 matrix. In addition, alpha3 integrin-null cells show low strength of adhesion to surfaces coated with purified laminin-5 compared to wild-type cells although both the wild type and the alpha3 integrin-null keratinocytes adhere equally strongly to laminin-5 that has been organized into arrays by other epithelial cells. These data suggest: (1) that alpha3beta1 integrin plays an important role in determining the incorporation of laminin-5 into its proper higher-order structure within the extracellular matrix of keratinocytes and (2) that the organizational state of laminin-5 has an influence on laminin-5 matrix function.     

Analysis of heterogeneity of human keratinocytes interacting with immobilized fibronectin and collagenes I and IV types

The concept that stem cells form an independent subpopulations of somatic cells assumes the heterogeneity of cellular populations in adult tissues. As skin keratinocytes have natural affinity to extracellular matrix proteins, we made an attempt to reveal subpopulations of these cells depending on the time of their adhesion to substrates of collagen I and IV types and fibronectin. After selection for 10, 20 and 30 min the keratinocytes were cultivated for 24 h. The area of cell projection on a substrate and the spreading coefficient were measured (Kuzminykh, Petrov, 2004; Petrov et al., 2007). In 24 h statistically reliable morphological differences between the cells depending on the substratum were found. The size of the cells growing on collagen I type was twice as large as that of the cells cultivated on collagen IV type or in fibronectin. Irrespective of the substratum, up to 60-65% of the cells had a rounded form. The cultivation on collagens revealed the heterogeneity of keratinocytes both in the control cultures and under selection by adhesion time, while the cells grown on fibronectin behaved as a homogeneous population. These results suggest that, contrary to fibronectin, collagens stabilize some physiological states of keratinocytes corresponding to their interaction with extracellular matrix proteins in the organism.     

Differences in the character of interaction of normal and transformed human keratinocytes with laminin isoforms

Laminins constitute a family of heterotrimeric glycoproteins of basement membranes. Laminins promote cell adhesion, migration, growth, and differentiation. So far, at least 12 different isoforms of laminin have been known. However, no sufficient knowledge is available on the nature of cell response on different laminins. The study was aimed to compare adhesive properties of two laminin isoforms, laminin-1 and laminin-2/4, with respect to normal (freshly isolated keratinocytes) and transformed (A-431) human skin cells. We have used the following assays: cell adhesion to the substrate covered with laminin isoformes, interaction of latex beads (D = 1 micron) coated with the same proteins with cells in suspension, and a comparative study of the cytoskeleton structure of cells spread on the immobilized laminins. It was demonstrated that laminin-2/4 is a more effective potent promotor of adhesion for both normal keratinocytes and transformed A-431 cells, compared with laminin-1. A comparison of many attached protein-covered beads allowed to estimate a relative quantity of cell surface receptors to laminin isoforms in different cell types. The relative number of receptors to laminin-2/4 on the keratinocyte surface is 7 times higher than that to laminin-1 after a 30 min incubation with cells, and is 6 times higher after 1 hour. As for A-431 cells, their attachment to laminin-2/4 beads is 5 times higher than that to laminin-1-beads after a 1 min incubation, but as early as after 5 min this distinction disappeared, owing to bead internalization. The presence of a specific receptor to laminin-2/4 but not to laminin-1 on the keratinocyte surface has been suggested. Keratin differences in cytoskeleton organization in normal and transformed skin cells spread on the substrates covered with laminin-1 and laminin-2/4 were demonstrated.     

Various effects of laminin-1 and laminin-2/4 on adhesion and migration of cultured human keratinocytes

The cell-matrix interaction is one of the factors defining the cell behavior in normal and wounded tissues. To determine the function of laminin-2/4, one of components of the skin basement membrane in the process of reepithelization, we studied its interaction with human keratinocytes. The adhesive properties of laminin-2/4 and its effect on keratinocytes migration in vitro were analysed. For comparison with our present investigation, we used the earlier studied laminin-1 from EHS mouse sarcoma. Laminin-2/4 appeared to be a good substrate for human keratinocytes, and this correlates with a greater number of cell surface receptors compared with laminin-1. Laminin-2/4 alone does not stimulate keratinocyte migration, but, in contrast to laminin-1, supports EGF-mediated migration. The obtained results give an insight into the function of laminin-2/4 in normal skin and during wound healing.     

Analysis of heterogeneity of human keratinocyte populations by their adhesion to substrate and keratin 19 to actin ratio

Keratin 19 is considered to be a marker for skin stem cells that assume a particular subpopulation of these cells in the keratinocyte population. It is also known that keratinocytes have a natural affinity for extracellular matrix proteins. The aim of this work was to identify subpopulations of keratinocytes by cultivation on various substrates (type-I collagen, laminin 2/4 and fibronectin) and by measuring keratin 19/actin ratio (G/R). The areas of cell projection on a substrate, perimeter, the spreading coefficient, and G/R have been assayed. Keratinocyte populations were morphologically homogeneous both on fibronectin and laminin 2/4. Large cells comprised 12 and 20% of the populations, respectively. No correlation between morphological parameters of cells and keratin 19/actin ratio was found in keratinocytes cultivated in fibronectin. The cells grown on collagen behaved as a heterogenous population with large cells comprising more than 50% of the population. On average, the size of the cells grown on collagen was twice as large as the cells cultivated on fibronectin. On the other hand, the correlation between cell size and G/R was revealed in cells grown on both collagen and laminin 2/4. The cells with lower G/R values display a larger size and higher spreading. We assume that this correlation is determined by the presence of α1 and α2 integrin subunits in these cells. Keratin 19 assay did not reveal keratinocyte subpopulations. Our results raised doubts regarding the presence of stem cells in cultures of humanskin keratinocytes.     

Ultrastructural and immunocytochemical detection of keratins and extracellular matrix proteins in lizard skin cultured in vitro

The present study shows the localization of epidermal and dermal proteins produced in lizard skin cultivated in vitro. Cells from the skin have been cultured for up to one month to detect the expression of keratins, actin, vimentin and extracellular matrix proteins (fibronectin, chondroitin sulphate proteoglycan, elastin and collagen I). Keratinocytes and dermal cells weakly immunoreact for Pan-Cytokeratin but not with the K17-antibody at the beginning of the cell culture when numerous keratin bundles are present in keratinocyte cytoplasm. The dense keratin network disappears after 7-12 days in culture, and K17 becomes detectable in both keratinocytes and mesenchymal cells isolated from the dermis. While most epidermal cells are lost after 2 weeks of in vitro cultivation dermal cells proliferate and form a pellicle of variable thickness made of 3-8 cell layers. The fibroblasts of this dermal equivalent produces an extracellular matrix containing chondroitin sulphate proteoglycan, collagen I, elastic fibers and fibronectin, explaining the attachment of the pellicle to the substratum. The study indicates that after improving keratinocyte survival a skin equivalent for lizard epidermis would be feasible as a useful tool to analyze the influence of the dermis on the process of epidermal differentiation and the control of the shedding cycle in squamates.     

Keratinocyte growth factor (KGF) promotes keratinocyte cell attachment and migration on collagen and fibronectin

Keratinocyte growth factor (KGF) induction of keratinocyte attachment and migration on provisional and basement membrane proteins was examined. KGF-treated keratinocytes showed increased attachment to collagen types I and IV and fibronectin, but, not to laminin-1, vitronectin, or tenascin. This increase was time- and dose-dependent. Increase in attachment occurred with 2 10 microg/ml of ECM proteins. This KGF-stimulated cell attachment was beta1 integrin-dependent but was not associated with stimulation of the cell surface expression nor affinity (activity) of the collagen integrin receptor (alpha2beta1) nor the fibronectin integrin receptors (alpha5beta1 or alphav). At the basal layer of KGF-treated cells significant accumulation of beta1 integrins was found at the leading edges, and actin stress fibers colocalized with beta1. KGF also induced migratory phenotype and stimulated keratinocyte migration on both fibronectin and collagen types I and IV but not on laminin-1, vitronectin nor tenascin. The results suggest that in addition to its proliferation promoting activity. KGF is able to modulate keratinocyte adhesion and migration on collagen and fibronectin. Our data suggest that KGF induced integrin avidity (clustering), a signaling event, which is not dependent on the alteration of cell surface integrin numbers.     

Adhesion of cultured bovine aortic endothelial cells to laminin-1 mediated by dystroglycan

Expression of dystroglycan (DG) by cultured bovine aortic endothelial (BAE) cells was confirmed by cDNA cloning from a BAE cDNA library, Northern blotting of mRNA, Western blotting of membrane proteins, and double immunostaining with antibodies against betaDG and platelet endothelial cell adhesion molecule-1. Immunocytochemical analysis revealed localization of DG in multiple plaques on the basal side of resting cells. This patchy distribution was obscured in migrating cells, in which the most prominent staining was observed in the trailing edge anchoring the cells to the substratum. Biotin-labeled laminin-1 overlay assay of dissociated BAE membrane proteins indicated the interaction of laminin-1 with alphaDG. The laminin alpha5 globular domain fragment expressed in bacteria and labeled with biotin could also bind alphaDG on the membrane blot, and the unlabeled fragment disrupted the binding of biotin-laminin-1 to alphaDG. The interaction of biotin-laminin-1 with alphaDG was inhibited by soluble alphaDG contained in the conditioned medium from DG cDNA-transfected BAE cells and by a series of glycosaminoglycans (heparin, dextran sulfate, and fucoidan). Soluble alphaDG in the conditioned medium inhibited the adhesion of BAE cells to laminin-1-coated dishes, whereas it had no effect on their adhesion to fibronectin. All three glycosaminoglycans that disrupted the biotin-laminin-1 binding to alphaDG inhibited BAE cell adhesion to laminin-1, whereas they failed to inhibit the adhesion to fibronectin. These results indicate a role of DG as a non-integrin laminin receptor involved in vascular endothelial cell adhesion to the extracellular matrix.     

Isolation, culture and identification of epidermal stem cells from newborn mouse skin

In healthy individuals, skin integrity is maintained by epidermal stem cells which self-renew and generate daughter cells that undergo terminal differentiation. Epidermal stem cells represent a promising source of stem cells, and their culture has great potential in scientific research and clinical application. However, no single method has been universally adopted for identifying and isolating epidermal stem cells. Here, we reported the isolation and characterization of putative epidermal stem cells from newborn mouse skin. The keratinocytes were separated enzymatically. Putative epidermal stem cells were selected by rapid adherence on a composite matrix made of type I collagen and fibronectin. Unattached cells were discarded after 10 min, and the attached cells were cultured in a defined culture medium. The isolated cells showed the typical epidermal stem cell morphology. Immunofluorescence indicated that the cells were strongly stained for β₁ integrin family of extracellular matrix receptors. In conclusion, mouse putative epidermal stem cells were successfully isolated from newborn mouse epidermis on the basis of high rapid adhesion to extracellular matrix proteins and cultured in vitro.     

Integrin beta4 regulates migratory behavior of keratinocytes by determining laminin-332 organization

Whether alpha6beta4 integrin regulates migration remains controversial. beta4 integrin-deficient (JEB) keratinocytes display aberrant migration in that they move in circles, a behavior that mirrors the circular arrays of laminin (LM)-332 in their matrix. In contrast, wild-type keratinocytes and JEB keratinocytes, induced to express beta4 integrin, assemble laminin-332 in linear tracks over which they migrate. Moreover, laminin-332-dependent migration of JEB keratinocytes along linear tracks is restored when cells are plated on wild-type keratinocyte matrix, whereas wild-type keratinocytes show rotation over circular arrays of laminn-332 in JEB keratinocyte matrix. The activities of Rac1 and the actin cytoskeleton-severing protein cofilin are low in JEB keratinocytes compared with wild-type cells but are rescued following expression of wild-type beta4 integrin in JEB cells. Additionally, in wild-type keratinocytes Rac1 is complexed with alpha6beta4 integrin. Moreover, Rac1 or cofilin inactivation induces wild-type keratinocytes to move in circles over rings of laminin-332 in their matrix. Together these data indicate that laminin-332 matrix organization is determined by the alpha6beta4 integrin/actin cytoskeleton via Rac1/cofilin signaling. Furthermore, our results imply that the organizational state of laminin-332 is a key determinant of the motility behavior of keratinocytes, an essential element of skin wound healing and the successful invasion of epidermal-derived tumor cells.     

Reconstitution of human epidermis in vitro is accompanied by transient activation of basal keratinocyte spreading

Frozen human cadaver skin obtained from the skin bank was thawed and incubated in serum-free medium for 1–2 days, after which the original epidermis could be removed mechanically. Transmission electron microscopic observations showed that the dermal matrix remaining behind contained intact bundles of collagen fibrils but no live cells and that a continuous lamina densa persisted in the basement membrane region. Indirect immunofluorescence analyses demonstrated linear staining of the basement membrane region by antibodies against laminin and type IV collagen and discontinuous staining with antibodies against fibronectin. Scanning electron microscopic observations revealed a normal topographical arrangement of dermal matrix papilla and interspersed crypts on the surface of the matrix. Epidermal cells placed on the dermal matrix attached in 1–2 h and spread by 24 h. After 1 week of culture the epidermis was reconstituted, at which time approximately 30% of the epidermal cells were basal keratinocytes and the remainder were more differentiated keratinocytes. A high degree of differentiation of the reconstituted epidermis was shown by the formation of hemidesmosomes along the basement membrane, the formation of desmosomes characterized by intercellular dense lines, and the presence of a cell layer containing keratohyalin granules. At various times during epidermal reconstitution, cells were harvested and tested in short-term assays for adhesion to fibronectin substrata. During the first several days there was a transient activation of basal keratinocyte spreading analogous to the modulation of keratinocyte spreading that we have observed during epidermal reconstitution in vivo.     

Type XVII collagen (BP180) can function as a cell−matrix adhesion molecule via binding to laminin 332

Collagen XVII (COL17) is a transmembrane glycoprotein that is expressed on the basal surface of basal epidermal keratinocytes. Previous observations have led to the hypothesis that an interaction between COL17 and laminin 332, an extracellular matrix protein, contributes to the attachment of the basal keratinocyte to the basement membrane. In order to isolate and manipulate COL17 interactions with ECM components, we induced COL17 expression in two cells lines, SK-MEL1 and K562, that exhibit little or no capacity to attach to our test substrates, including laminin 332, types I and IV collagen, and fibronectin. Cells expressing high levels of COL17 preferentially adhered to a laminin 332 matrix, and, to a lesser extent, type IV collagen, while showing little or no binding to type I collagen or fibronectin. A quantitative analysis of cell adhesive forces revealed that, compared with COL17-negative cells, COL17-positive cells required over 7-fold greater force to achieve 50% detachment from a laminin 332 substrate. When a cell preparation (either K562 or SK-MEL1) with heterogeneous COL17 expression levels was allowed to attach to a laminin 332 matrix, the COL17-positive and COL17-negative cells differentially sorted to the bound and unbound cell fractions, respectively. COL17-dependent attachment to laminin 332 could be reduced or abolished by siRNA-mediated knock-down of COL17 expression or by adding to the assay wells specific antibodies against COL17 or laminin 332. These findings provide strong support for the hypothesis that cell surface COL17 can interact with laminin 332 and, together, participate in the adherence of a cell to the extracellular matrix.     

Keratinocyte-derived laminin-332 promotes adhesion and migration in melanocytes and melanoma

Melanocytes are highly motile cells that play an integral role in basic skin physiological processes such as wound healing and proper skin pigmentation. It has been postulated that surrounding keratinocytes contribute to melanocyte migration, but underlying mechanisms remain rather vague so far. In this study, we set out to analyze the specific potential contribution of keratinocyte components to melanocytes and melanoma cell migration-related processes. Our studies revealed that A375 human melanoma cell attachment, spreading, and migration are interestingly better supported by HaCaT keratinocyte extracellular matrix (ECM) than by self-derived A375 ECM. Moreover, HaCaT ECM caused increased integrin α6 expression, adhesion-mediated focal adhesion kinase phosphorylation, and focal adhesion formations. Similar effects were confirmed in human melanocytes. Furthermore, we found that keratinocyte-derived soluble factors did not appear to significantly contribute to these processes. Specific extrinsic factors that promoted melanoma migration were attributed to keratinocyte-derived laminin-332, whereas alternative ECM component such as laminin-111 and fibronectin functions appeared to have insignificant contributions. Taken together, these studies implicate extrinsic laminin-332 in promoting the high mobility property and perhaps invasiveness inherently characteristic of, and that are the menace of, melanocytes and melanomas, respectively.