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.     

Distinct Developmental Functions of Prostasin (CAP1/PRSS8) Zymogen and Activated Prostasin

The membrane-anchored serine prostasin (CAP1/PRSS8) is essential for barrier acquisition of the interfollicular epidermis and for normal hair follicle development. Consequently, prostasin null mice die shortly after birth. Prostasin is found in two forms in the epidermis: a one-chain zymogen and a two-chain proteolytically active form, generated by matriptase-dependent activation site cleavage. Here we used gene editing to generate mice expressing only activation site cleavage-resistant (zymogen-locked) endogenous prostasin. Interestingly, these mutant mice displayed normal interfollicular epidermal development and postnatal survival, but had defects in whisker and pelage hair formation. These findings identify two distinct in vivo functions of epidermal prostasin: a function in the interfollicular epidermis, not requiring activation site cleavage, that can be mediated by the zymogen-locked version of prostasin and a proteolysis-dependent function of activated prostasin in hair follicles, dependent on zymogen conversion by matriptase.     

TMEM45A Is Dispensable for Epidermal Morphogenesis,Keratinization and Barrier Formation

TMEM45A gene encodes an initially uncharacterized predicted transmembrane protein. We previously showed that this gene is highly expressed in keratinocytes where its expression correlates with keratinization, suggesting a role in normal epidermal physiology. To test this hypothesis, we generated TMEM45A knockout mice and found that these mice develop without any evident phenotype. The morphology of the epidermis assessed by histology and by labelling differentiation markers in immunofluorescence was not altered. Toluidine blue permeability assay showed that the epidermal barrier develops normally during embryonic development. We also showed that depletion of TMEM45A in human keratinocytes does not alter their potential to form in vitro 3D-reconstructed epidermis. Indeed, epidermis with normal morphogenesis were generated from TMEM45A-silenced keratinocytes. Their expression of differentiation markers quantified by RT-qPCR and evidenced by immunofluorescence labelling as well as their barrier function estimated by Lucifer yellow permeability were similar to the control epidermis. In summary, TMEM45A gene expression is dispensable for epidermal morphogenesis, keratinization and barrier formation. If this protein plays a role in the epidermis, its experimental depletion can possibly be compensated by other proteins in the two experimental models analyzed in this study.     

Impaired hair follicle morphogenesis and polarized keratinocyte movement upon conditional inactivation of integrin-linked kinase in the epidermis

Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements, two key aspects of epithelial morphogenesis and function.     

Multiple classes of stem cells in cutaneous epithelium: a lineage analysis of adult mouse skin

Continuous renewal of the epidermis and its appendages throughout life depends on the proliferation of a distinct population of cells called stem cells. We have used in situ retrovirus-mediated gene transfer to genetically mark cutaneous epithelial stem cells of adolescent mice, and have followed the fate of the marked progeny after at least 37 epidermal turnovers and five cycles of depilation-induced hair growth. Histological examination of serial sections of labeled pilosebaceous units demonstrated a complex cell lineage. In most instances, labeled cells were confined to one or more follicular compartments or solely to sebaceous glands. Labeled keratinocytes in interfollicular epidermis were confined to distinct columnar units representing epidermal proliferative units. The contribution of hair follicles to the epidermis was limited to a small rim of epidermis at the margin of the follicle, indicating that long term maintenance of interfollicular epidermis was independent of follicle-derived cells. Our results indicate the presence of multiple stem cells in cutaneous epithelium, some with restricted lineages in the absence of major injury.     

Combined Inhibition of IGF-1R/IR and Src Family Kinases Enhances Antitumor Effects in Prostate Cancer by Decreasing Activated Survival Pathways

Background

Treatment of metastatic prostate cancer (PCa) with single agents has shown only modest efficacy. We hypothesized dual inhibition of different pathways in PCa results in improved tumor inhibition. The Src family kinases (SFK) and insulin-like growth factor-1 (IGF-1) signaling axes are aberrantly activated in both primary PCa and bone metastases and regulate distinct and overlapping functions in PCa progression. We examined the antitumor effects of combined inhibition of these pathways.

Materials and Methods

Src andIGF-1 receptor (IGF-1R) inhibition was achieved in vitro by short hairpin (sh)RNA and in vitro and in vivo by small molecule inhibitors (dasatinib and BMS-754807, against SFK and IGF-1R/Insulin Receptor(IR), respectively).

Results

In vitro, inhibition of IGF-1 signaling affected cell survival and proliferation. SFK blockade alone had modest effects on proliferation, but significantly enhanced the IGF-1R blockade. These findings correlated with a robust inhibition of IGF-1-induced Akt1 phophorylation by dasatinib, whereas Akt2 phosphorylation was SFK independent and only inhibited by BMS-754807. Thus, complete inhibition of both Akt genes, not seen by either drug alone, is likely a major mechanism for the decreased survival of PCa cells. Furthermore, dasatinib and BMS-754807 inhibited in vivo growth of the primary human xenograft MDA PCa 133, with corresponding inhibition of Akt in tumors. Also, both orthotopic and intratibial tumor growth of PC-3 cells were more potently inhibited by dual SFK and IGF-1R/IR blockade compared to either pathway alone, with a corresponding decrease in bone turnover markers.

Conclusions

Dual IGF-1R/IR and SFK inhibition may be a rational therapeutic approach in PCa by blocking both independent and complementary processes critical to tumor growth.     

Enhanced contact allergen- and UVB-induced keratinocyte apoptosis in the absence of CD95/Fas/Apo-1

FAS/CD95/Apo-1 is a ubiquitously expressed cell-surface receptor involved in the initiation of programmed cell death. Its function in epidermal keratinocytes has been incompletely defined. Available evidence from in vitro studies points to important roles of Fas in the pathogenesis of contact dermatitis and in keratinocyte apoptosis induced by ultraviolet light. To define functions of Fas in the epidermis in vivo, we have generated mice with epidermis-specific deletion of the fas gene and tested its requirement for 2,4-dinitrofluorobenzene-induced contact dermatitis and for ultraviolet light B (UVB)-induced keratinocyte apoptosis. We report here our unexpected finding that keratinocyte apoptosis induced by both a contact allergen and UVB irradiation was significantly enhanced in Fas-negative epidermis. Expression of Fas by epidermal keratinocytes was neither necessary for the normal development of contact hypersensitivity of the skin, nor required for keratinocyte apoptosis following UVB irradiation. Our study results thus show that in the epidermis in vivo Fas exerts antiapoptotic effects that outweigh its proapoptotic role in contact hypersensitivity responses of the skin and in the tissue response of the epidermis to UVB irradiation.     

UVB-induced senescence in human keratinocytes requires a functional insulin-like growth factor-1 receptor and p53

To cope with the frequent exposure to carcinogenic UV B (UVB) wavelengths found in sunlight, keratinocytes have acquired extensive protective measures to handle UVB-induced DNA damage. Recent in vitro and epidemiological data suggest one these protective mechanisms is dependent on the functional status of the insulin-like growth factor-1 receptor (IGF-1R) signaling network in keratinocytes. During the normal UVB response, ligand-activated IGF-1Rs protect keratinocytes from UVB-induced apoptosis; however, as a consequence, these keratinocytes fail to proliferate. This adaptive response of keratinocytes to UVB exposure maintains the protective barrier function of the epidermis while ensuring that UVB-damaged keratinocytes do not replicate DNA mutations. In contrast, when keratinocytes are exposed to UVB in the absence of IGF-1R activation, the keratinocytes are more sensitive to UVB-induced apoptosis, but the surviving keratinocytes retain the capacity to proliferate. This aberrant UVB response represents flawed protection from UVB damage potentially resulting in the malignant transformation of keratinocytes. Using normal human keratinocytes grown in vitro, we have demonstrated that activation of the IGF-1R promotes the premature senescence of UVB-irradiated keratinocytes through increased generation of reactive oxygen species (ROS) and by maintaining the expression of the cyclin-dependent kinase inhibitor p21^CDKN1A. Furthermore, IGF-1R–dependent UVB-induced premature senescence required the phosphorylation of p53 serine 46. These data suggest one mechanism of keratinocyte resistance to UVB-induced carcinogenesis involves the induction of IGF-1R–dependent premature senescence.     

Beta-catenin and Hedgehog signal strength can specify number and location of hair follicles in adult epidermis without recruitment of bulge stem cells

Using K14deltaNbeta-cateninER transgenic mice, we show that short-term, low-level beta-catenin activation stimulates de novo hair follicle formation from sebaceous glands and interfollicular epidermis, while only sustained, high-level activation induces new follicles from preexisting follicles. The Hedgehog pathway is upregulated by beta-catenin activation, and inhibition of Hedgehog signaling converts the low beta-catenin phenotype to wild-type epidermis and the high phenotype to low. beta-catenin-induced follicles contain clonogenic keratinocytes that express bulge markers; the follicles induce dermal papillae and provide a niche for melanocytes, and they undergo 4OHT-dependent cycles of growth and regression. New follicles induced in interfollicular epidermis are derived from that cellular compartment and not through bulge stem cell migration or division. These results demonstrate the remarkable capacity of adult epidermis to be reprogrammed by titrating beta-catenin and Hedgehog signal strength and establish that cells from interfollicular epidermis can acquire certain characteristics of bulge stem cells.     

Deconstructing the skin: cytoarchitectural determinants of epidermal morphogenesis

To provide a stable environmental barrier, the epidermis requires an integrated network of cytoskeletal elements and cellular junctions. Nevertheless, the epidermis ranks among the body's most dynamic tissues, continually regenerating itself and responding to cutaneous insults. As keratinocytes journey from the basal compartment towards the cornified layers, they completely reorganize their adhesive junctions and cytoskeleton. These architectural components are more than just rivets and scaffolds - they are active participants in epidermal morphogenesis that regulate epidermal polarization, signalling and barrier formation.     

CD98, a novel marker of transient amplifying human keratinocytes

Identification of plasma membrane markers of basal keratinocytes is essential for sorting basal cells and, subsequently, adult epidermal stem cells. In this study, we isolated caveolin-1-enriched microdomains from human HaCaT keratinocytes and identified proteins representing potential cell surface markers of the epidermis by a proteomic approach. The purification of this caveolae domain allowed us to characterize 53 proteins of which 26% were transmembrane and 32% associated-membrane proteins. One of them, CD98, was found to be co-localized with beta1 integrin at the plasma membrane of the basal keratinocytes of healthy human epidermis. We then isolated CD98-positive keratinocytes from fresh skin biopsies. Using clonogenic assays, we demonstrate that CD98 may be considered as a marker of transient amplifying human keratinocytes.     

Rac1 is crucial for hair follicle integrity but is not essential for maintenance of the epidermis

Rac1 is a small GTPase that regulates the actin cytoskeleton but also other cellular processes. To investigate the function of Rac1 in skin, we generated mice with a keratinocyte-restricted deletion of the rac1 gene. Rac1-deficient mice lost nearly all of their hair within a few weeks after birth. The nonpermanent part of mutant hair follicles developed constrictions; lost expression of hair follicle-specific keratins, E-cadherin, and alpha6 integrin; and was eventually removed by macrophages. The permanent part of hair follicles and the sebaceous glands were maintained, but no regrowth of full-length hair follicles was observed. In the skin of mutant mice, epidermal keratinocytes showed normal differentiation, proliferation, cell-cell contacts, and basement membrane deposition, demonstrating no obvious defects of Rac1-deficient epidermis in vivo. In vitro, Rac1-null keratinocytes displayed a strong spreading defect and slightly impaired adhesion. These data show that Rac1 plays an important role in sustaining the integrity of the lower part of hair follicles but not in maintenance of the epidermis.     

UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph Polarize F-Actin during Embryonic Morphogenesis by Regulating the WAVE/SCAR Actin Nucleation Complex

Many cells in a developing embryo, including neurons and their axons and growth cones, must integrate multiple guidance cues to undergo directed growth and migration. The UNC-6/netrin, SLT-1/slit, and VAB-2/Ephrin guidance cues, and their receptors, UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph, are known to be major regulators of cellular growth and migration. One important area of research is identifying the molecules that interpret this guidance information downstream of the guidance receptors to reorganize the actin cytoskeleton. However, how guidance cues regulate the actin cytoskeleton is not well understood. We report here that UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph differentially regulate the abundance and subcellular localization of the WAVE/SCAR actin nucleation complex and its activator, Rac1/CED-10, in the Caenorhabditis elegans embryonic epidermis. Loss of any of these three pathways results in embryos that fail embryonic morphogenesis. Similar defects in epidermal enclosure have been observed when CED-10/Rac1 or the WAVE/SCAR actin nucleation complex are missing during embryonic development in C. elegans. Genetic and molecular experiments demonstrate that in fact, these three axonal guidance proteins differentially regulate the levels and membrane enrichment of the WAVE/SCAR complex and its activator, Rac1/CED-10, in the epidermis. Live imaging of filamentous actin (F-actin) in embryos developing in the absence of individual guidance receptors shows that high levels of F-actin are not essential for polarized cell migrations, but that properly polarized distribution of F-actin is essential. These results suggest that proper membrane recruitment and activation of CED-10/Rac1 and of WAVE/SCAR by signals at the plasma membrane result in polarized F-actin that permits directed movements and suggest how multiple guidance cues can result in distinct changes in actin nucleation during morphogenesis.