Role of melanoma chondroitin sulphate proteoglycan in patterning stem cells in human interfollicular epidermis

Human interfollicular epidermis is renewed by stem cells that are clustered in the basal layer in a patterned, non-random distribution. Stem cells can be distinguished from other keratinocytes by high expression of beta1 integrins and lack of expression of terminal differentiation markers; they divide infrequently in vivo but form actively growing colonies in culture. In a search for additional stem cell markers, we observed heterogeneous epidermal expression of melanoma chondroitin sulphate proteoglycan (MCSP). MCSP was expressed by those keratinocytes with the highest beta1 integrin levels. In interfollicular epidermis, expression was confined to non-cycling cells and, in culture, to self-renewing clones. However, fluorescence-activated cell sorting on the basis of MCSP and beta1 integrin expression gave no more enrichment for clonogenic keratinocytes than sorting for beta1 integrins alone. To interfere with endogenous MCSP, we retrovirally infected keratinocytes with a chimera of the CD8 extracellular domain and the MCSP cytoplasmic domain. CD8/MCSP did not affect keratinocyte proliferation or differentiation but the cohesiveness of keratinocytes in isolated clones or reconstituted epidermal sheets was greatly reduced. CD8/MCSP caused stem cell progeny to scatter without differentiating. CD8/MCSP did not alter keratinocyte motility but disturbed cadherin-mediated cell-cell adhesion and the cortical actin cytoskeleton, effects that could be mimicked by inhibiting Rho. We conclude that MCSP is a novel marker for epidermal stem cells that contributes to their patterned distribution by promoting stem cell clustering.     

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.     

The tetraspanin CD9 associates with the integrin alpha6beta4 in cultured human epidermal keratinocytes and is involved in cell motility

Integrins are involved in several ways in keratinocyte physiology, including cell motility. CD9 is a member of the tetraspanin protein family which is found in association with other transmembrane proteins like the integrins. CD9 is expressed in the epidermal tissue, but this expression is not regulated by differentiation. The present work focuses on association of CD9 with the integrin alpha6beta4 in keratinocytes. In vivo, CD9 does not co-localize with alpha6beta4, and is not internalized with the integrin upon basal detachment with dispase. In vitro, CD9 is found partly in co-localization with alpha6beta4 and is internalized with the integrin after keratinocyte detachment with dispase. Using blocking antibodies in a phagokinetic tracks assay, we show that CD9, and to a lesser extent alpha6beta4, but not the tetraspanin CD82, promote motility of subconfluent keratinocytes on collagen I. Our observations also suggest that CD9 is involved in the formation of lamellipodia. We also report that the phorbol ester TPA has no effect on CD9 expression and association with alpha6beta4, but increases keratinocyte motility, possibly through modulation of integrin subunits expression, or through upregulation of collagenase-1 expression. Together, these results confirm that CD9 associates with alpha6beta4 in cultured keratinocytes, possibly in order to regulate the function of the integrin, and that CD9 is involved in keratinocyte motility on collagen. The data suggest that regulation of adhesion characteristics by CD9 in keratinocytes may play a role in epidermal repair.     

Myc,Cdk2 and cellular senescence: Old players,new game

The aberrant activation of oncogenic pathways promotes tumor progression, but concomitantly elicits compensatory tumor-suppressive responses, such as apoptosis or senescence. For example, Ras induces senescence, while Myc generally triggers apoptosis. Myc is in fact viewed as an anti-senescence oncogene, as it is a potent inducer of cell proliferation and immortalization, bypasses growth-inhibitory signals, and cooperates with Ras in cellular transformation. Recent reports prompt re-evaluation of Myc-induced senescence, and of its role in tumor progression and therapy. We have shown that the cyclin-dependent kinase Cdk2, although redundant for cell cycle progression, has a unique role in suppressing a Myc-induced senescence program: Myc activation elicited expression of p16^INK4a and p21^Cip1, and caused senescence in cell lacking Cdk2, but not in Cdk2-proficient cells. Additional cellular activities have been identified that suppress Myc-induced senescence, including the Wrn helicase, Telomerase and Miz1. These senescence-suppressing activities were critical for tumor progression, as deficiency in Cdk2, telomerase or Miz1 reduced the onset of Myc-induced lymphoma in transgenic mice. Other gene products like p53, SUV39H1 or TGFß promoted senescence, which together with apoptosis contributed to tumor suppression. Paradoxically, Myc directly counteracted the very same senescence program that it potentially elicits, since it positively regulated Wrn, Telomerase and Cdk2 activity, and Cdk2 inhibition re-activated the latent senescence program in Myc expressing cells. Hence, while these molecules are instrumental to the oncogenic action of Myc, they may simultaneously constitute its Achille's heel for therapeutic development.     

The ubiquitin ligase HectH9 regulates transcriptional activation by Myc and is essential for tumor cell proliferation

The Myc oncoprotein forms a binary activating complex with its partner protein, Max, and a ternary repressive complex that, in addition to Max, contains the zinc finger protein Miz1. Here we show that the E3 ubiquitin ligase HectH9 ubiquitinates Myc in vivo and in vitro, forming a lysine 63-linked polyubiquitin chain. Miz1 inhibits this ubiquitination. HectH9-mediated ubiquitination of Myc is required for transactivation of multiple target genes, recruitment of the coactivator p300, and induction of cell proliferation by Myc. HectH9 is overexpressed in multiple human tumors and is essential for proliferation of a subset of tumor cells. Our results suggest that site-specific ubiquitination regulates the switch between an activating and a repressive state of the Myc protein, and they suggest a strategy to interfere with Myc function in vivo.     

Changes in keratinocyte adhesion during terminal differentiation: reduction in fibronectin binding precedes alpha 5 beta 1 integrin loss from the cell surface

During terminal differentiation keratinocytes move out of the basal layer of the epidermis and thereby lose contact with the basement membrane. We show that terminal differentiation in culture involves loss of adhesiveness to fibronectin, laminin, and collagen types I and IV. The adhesive changes precede, by several hours, loss of the alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1 integrins from the cell surface. Keratinocyte adhesion to fibronectin is mediated by the alpha 5 beta 1 integrin, and the decrease in adhesion of intact cells to fibronectin is correlated with a decrease in the ability of alpha 5 beta 1 receptors to bind fibronectin. Thus modulation of integrin function early in terminal differentiation may be an early event determining cell migration out of the basal layer.     

The control of polarized integrin topography and the organization of adhesion-related cytoskeleton in normal human keratinocytes depend upon number of passages in culture and ionic environment.

Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are mediated, besides other molecules, by integrin receptors that are sorted to defined membrane domains. The hemidesmosome-associated integrin alpha 6 beta 4 is sharply localized to the basal surface of basal cells while alpha 2 beta 1 and alpha 3 beta 1 are enriched laterally. This integrin sorting pattern is perfectly reproducible in vitro by cultured keratinocytes and takes place progressively in primary or secondary culture in the presence of 1.8 mM Ca2+. The polarized topography of integrins is gradually lost with higher passage numbers and between passage 5 and passage 7 there is a complete pericellular redistribution of the above integrins. Along with the decreased basal adhesive value of alpha 6 beta 4 there is a marked increase in the number of focal contacts in high-passage keratinocyte colonies. A similar loss of polarized topography of integrins occurs under low-Ca2+ culture conditions. Increasing the number of culture passages beyond the fifth induces the appearance of the fibronectin receptor alpha 5 beta 1 on the surface of keratinocytes, particularly at intercellular junctions and in some focal contacts. The receptor alpha 5 beta 1 is not detectably exposed by low-passage cells. We propose that forcing keratinocytes into more frequent cell cycles by continuous passaging may perturb the polarized topography of integrins and the adhesion mechanisms of keratinocytes. Then, low-passage keratinocytes are, in our opinion, the most reliable in vitro models for studying the physiology of epidermal cells.     

Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human epidermal keratinocytes and non-differentiating keratinocytes

We have compared the adhesive properties and integrin expression profiles of cultured human epidermal keratinocytes and a strain of nondifferentiating keratinocytes (ndk). Both cell types adhered to fibronectin, laminin, and collagen types I and IV, but ndk adhered more rapidly and at lower coating concentrations of the proteins. Antibody blocking experiments showed that adhesion of both cell types to fibronectin was mediated by the alpha 5 beta 1 integrin and to laminin by alpha 3 beta 1 in synergy with alpha 2 beta 1. Keratinocytes adhered to collagen with alpha 2 beta 1, but an antibody to alpha 2 did not inhibit adhesion of ndk to collagen. Both cell types adhered to vitronectin by alpha v-containing integrins. Immunoprecipitation of surface-iodinated and metabolically labeled cells showed that in addition to alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1, both keratinocytes and ndk expressed alpha 6 beta 4 and alpha v beta 5. ndk expressed all these integrins at higher levels than normal keratinocytes. ndk, but not normal keratinocytes, expressed alpha v beta 1 and alpha v beta 3; they also expressed alpha 1 beta 1, an integrin that was not consistently detected on normal keratinocytes. Immunofluorescence experiments showed that in stratified cultures of normal keratinocytes integrin expression was confined to cells in the basal layer; terminally differentiating cells were unstained. In contrast, all cells in the ndk population were integrin positive. Our observations showed that the adhesive properties of ndk differ from normal keratinocytes and reflect differences in the type of integrins expressed, the level of expression and the distribution of integrins on the cell surface. ndk thus have a number of characteristics that distinguish them from normal basal keratinocytes.     

Interaction of integrins alpha 3 beta 1 and alpha 2 beta 1: potential role in keratinocyte intercellular adhesion

The colocalization of integrins alpha 2 beta 1 and alpha 3 beta 1 at intercellular contact sites of keratinocytes in culture and in epidermis suggests that these integrins may mediate intercellular adhesion (ICA). P1B5, an anti-alpha 3 beta 1 mAb previously reported to inhibit keratinocyte adhesion to epiligrin, was also found to induce ICA. Evidence that P1B5-induced ICA was mediated by alpha 2 beta 1 and alpha 3 beta 1 was obtained using both ICA assays and assays with purified, mAb-immobilized integrins. Selective binding of alpha 2 beta 1-coated beads to epidermal cells or plate-bound alpha 3 beta 1 was observed. This binding was inhibited by mAbs to integrin alpha 3, alpha 2, or beta 1 subunits and could be stimulated by P1B5. We also demonstrate a selective and inhibitable interaction between affinity- purified integrins alpha 2 beta 1 and alpha 3 beta 1. Finally, we show that expression of alpha 2 beta 1 by CHO fibroblasts results in the acquisition of collagen and alpha 3 beta 1 binding. Binding to both of these ligands is inhibited by P1H5, an anti-alpha 2 beta 1 specific mAb. Results of these in vitro experiments suggest that integrins alpha 2 beta 1 and alpha 3 beta 1 can interact and may do so to mediate ICA in vivo. Thus, alpha 3 beta 1 mediates keratinocyte adhesion to epiligrin and plays a second role in ICA via alpha 2 beta 1.     

The involvement of Gab1 and PI 3-kinase in beta1 integrin signaling in keratinocytes

The control of the stem cell compartment in epidermis is closely linked to the regulation of keratinocyte proliferation and differentiation. Beta1 integrins are expressed 2-fold higher by stem cells than transit-amplifying cells. Signaling from these beta1 integrins is critical for the regulation of the epidermal stem cell compartment. To clarify the functional relevance of this differential expression of beta1 integrins, we established HaCaT cells with high beta1 integrin expression by repeated flow cytometric sorting of this population from the parental cell line. In these obtained cells expressing beta1 integrins by 5-fold, MAPK activation was markedly increased. Regarding the upstream of MAPK, Gab1 phosphorylation was also higher with high beta1 integrin expression, while Shc phosphorylation was not altered. In addition, enhanced phosphatidylinositol 3-kinase activation was also observed. These observations suggest that Gab1 and phosphatidylinositol 3-kinase play pivotal roles in the beta1 integrin-mediated regulation of the epidermal stem cell compartment.     

Role of LIM kinases in normal and psoriatic human epidermis

We present evidence that LIM kinases can control cell adhesion and compaction in human epidermis. LIMK2 is expressed in the epidermal basal layer and signals downstream of the GTPase Rac1 to promote extracellular matrix adhesion and inhibit terminal differentiation. Conversely, LIMK1 is expressed in the upper granular layers and phosphorylates and inhibits cofilin. Expression of LIMK1 is lost in psoriatic lesions and other skin disorders characterized by lack of cell compaction in the differentiating cell layers. In psoriatic lesions down-regulation of LIMK1 correlates with up-regulation of Myc. Expression of constitutively active cofilin or Myc in reconstituted human epidermis blocks cell compaction. Overexpression of LIMK1 leads to down-regulation of Myc, whereas inhibition of Rho kinase, an upstream activator of LIMK1, stimulates Myc expression. Inhibition of Myc by LIMK1 is via inhibition of Stat3 phosphorylation, because constitutively active cofilin or inhibition of Rho kinase results in Stat3 phosphorylation and increased Myc levels, whereas dominant negative Stat3 abolishes the effect. In conclusion, we have uncovered a novel antagonistic relationship between the LIMK1/phosphocofilin and Myc/Stat3 pathways in the differentiating layers of human epidermis and propose that down-regulation of LIMK1 contributes to one of the pathological features of psoriatic epidermal lesions.     

A novel muscle-specific beta 1 integrin binding protein (MIBP) that modulates myogenic differentiation

Myogenesis is regulated by cell adhesion receptors, including integrins of the beta1 family. We report the identification of a novel muscle-specific beta1 integrin binding protein (MIBP). MIBP binds to the membrane-proximal cytoplasmic region shared by beta1A and beta1D integrins, and the binding occurs in vivo as well as in vitro. Furthermore, we show that MIBP is abundantly expressed by C2C12 myogenic cells before fusion, and the expression of MIBP is dramatically downregulated during subsequent differentiation. Finally, we show that overexpression of MIBP in C2C12 cells resulted in a suppression of fusion and terminal differentiation, suggesting that MIBP may play a key role in controlling the progression of muscle differentiation.     

Desmoglein 1–dependent suppression of EGFR signaling promotes epidermal differentiation and morphogenesis

Dsg1 (desmoglein 1) is a member of the cadherin family of Ca²⁺-dependent cell adhesion molecules that is first expressed in the epidermis as keratinocytes transit out of the basal layer and becomes concentrated in the uppermost cell layers of this stratified epithelium. In this study, we show that Dsg1 is not only required for maintaining epidermal tissue integrity in the superficial layers but also supports keratinocyte differentiation and suprabasal morphogenesis. Dsg1 lacking N-terminal ectodomain residues required for adhesion remained capable of promoting keratinocyte differentiation. Moreover, this capability did not depend on cytodomain interactions with the armadillo protein plakoglobin or coexpression of its companion suprabasal cadherin, Dsc1 (desmocollin 1). Instead, Dsg1 was required for suppression of epidermal growth factor receptor–Erk1/2 (extracellular signal-regulated kinase 1/2) signaling, thereby facilitating keratinocyte progression through a terminal differentiation program. In addition to serving as a rigid anchor between adjacent cells, this study implicates desmosomal cadherins as key components of a signaling axis governing epithelial morphogenesis.