WIKI4, a Novel Inhibitor of Tankyrase and Wnt/?-Catenin Signaling

The Wnt/ß-catenin signaling pathway controls important cellular events during development and often contributes to disease when dysregulated. Using high throughput screening we have identified a new small molecule inhibitor of Wnt/ß-catenin signaling, WIKI4. WIKI4 inhibits expression of ß-catenin target genes and cellular responses to Wnt/ß-catenin signaling in cancer cell lines as well as in human embryonic stem cells. Furthermore, we demonstrate that WIKI4 mediates its effects on Wnt/ß-catenin signaling by inhibiting the enzymatic activity of TNKS2, a regulator of AXIN ubiquitylation and degradation. While TNKS has previously been shown to be the target of small molecule inhibitors of Wnt/ß-catenin signaling, WIKI4 is structurally distinct from previously identified TNKS inhibitors.     

Keratinocyte-Targeted Expression of Human Laminin γ2 Rescues Skin Blistering and Early Lethality of Laminin γ2 Deficient Mice

Laminin-332 is a heterotrimeric basement membrane component comprised of the α3, ß3, and γ2 laminin chains. Laminin-332 modulates epithelial cell processes, such as adhesion, migration, and differentiation and is prominent in many embryonic and adult tissues. In skin, laminin-332 is secreted by keratinocytes and is a key component of hemidesmosomes connecting the keratinocytes to the underlying dermis. In mice, lack of expression of any of the three Laminin-332 chains result in impaired anchorage and detachment of the epidermis, similar to that seen in human junctional epidermolysis bullosa, and death occurs within a few days after birth. To bypass the early lethality of laminin-332 deficiency caused by the knockout of the mouse laminin γ2 chain, we expressed a dox-controllable human laminin γ2 transgene under a keratinocyte-specific promoter on the laminin γ2 (Lamc2) knockout background. These mice appear similar to their wild-type littermates, do not develop skin blisters, are fertile, and survive >1.5 years. Immunofluorescence analyses of the skin showed that human laminin γ2 colocalized with mouse laminin α3 and ß3 in the basement membrane zone underlying the epidermis. Furthermore, the presence of “humanized” laminin-332 in the epidermal basement membrane zone rescued the alterations in the deposition of hemidesmosomal components, such as plectin, collagen type XVII/BP180, and integrin α6 and ß4 chains, seen in conventional Lamc2 knockout mice, leading to restored formation of hemidesmosomes. These mice will be a valuable tool for studies of organs deficient in laminin-332 and the role of laminin-332 in skin, including wound healing.     

Localization and regulation of epidermal 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity by barrier requirements

Recent studies have shown that epidermal cholesterol synthesis is regulated by HMG CoA reductase activity and that this activity is modulated by changes in the cutaneous permeability barrier. Here, we quantitated HMG CoA reductase activity after acute and chronic barrier disruption in the upper and lower layers of murine epidermis. In unperturbed epidermis, 13 and 87% of enzyme activity localized to the upper and lower epidermis, respectively, with the majority of activity in the stratum basale. Acute barrier disruption with either acetone or sodium dodecylsulfate provoked an increase in HMG CoA reductase activity (54% and 30%) in the lower layers, but only a small change in the upper layers. However, the activation state of the enzyme was increased 50% in the upper epidermis. Correction of barrier function by occlusion with an impermeable Latex wrap prevented the increase both in enzyme activity and activation state. After chronic barrier disruption; i.e., essential fatty acid deficient (EFAD) diet, HMG CoA reductase activity was increased in the upper epidermis (161%); a change prevented by occlusion. These results show: (1) that HMG CoA reductase activity is present in both the upper and lower cell layers; (2) that acute insults to barrier integrity stimulate enzyme activity in both the upper and lower epidermis; and (3) that chronic insults provoke an increase in enzyme activity in the upper layers. These studies provide further insights into the linkage of the permeability barrier with epidermal cholesterol metabolism.     

Towards a Quantitative Theory of Epidermal Calcium Profile Formation in Unwounded Skin

We propose and mathematically examine a theory of calcium profile formation in unwounded mammalian epidermis based on: changes in keratinocyte proliferation, fluid and calcium exchange with the extracellular fluid during these cells’ passage through the epidermal sublayers, and the barrier functions of both the stratum corneum and tight junctions localised in the stratum granulosum. Using this theory, we develop a mathematical model that predicts epidermal sublayer transit times, partitioning of the epidermal calcium gradient between intracellular and extracellular domains, and the permeability of the tight junction barrier to calcium ions. Comparison of our model’s predictions of epidermal transit times with experimental data indicates that keratinocytes lose at least 87% of their volume during their disintegration to become corneocytes. Intracellular calcium is suggested as the main contributor to the epidermal calcium gradient, with its distribution actively regulated by a phenotypic switch in calcium exchange between keratinocytes and extracellular fluid present at the boundary between the stratum spinosum and the stratum granulosum. Formation of the extracellular calcium distribution, which rises in concentration through the stratum granulosum towards the skin surface, is attributed to a tight junction barrier in this sublayer possessing permeability to calcium ions that is less than 15 nm s⁻¹ in human epidermis and less than 37 nm s⁻¹ in murine epidermis. Future experimental work may refine the presented theory and reduce the mathematical uncertainty present in the model predictions.     

Epidermal keratinocytes: regulation of multiple cell phenotypes by multiple protein kinase C isoforms

Squamous cells form the outermost layers of the epidermis, and though they are readily discarded from the tissue, they serve a vital water barrier function while in the stratum corneum. The generation of cornified or squamous keratinocytes involves a complex, multi-step differentiation process that insures the proper physical and immunological barrier functions of the epidermis are maintained. The regulation of keratinocyte terminal differentiation is influenced by a large number of signaling pathways. This article will review some recent findings regarding the roles of the protein kinase C (PKC) family in normal keratinocyte differentiation, as well as their involvement in skin diseases, especially skin cancer.     

Expression of NADH-dependent glutamate synthase protein in the epidermis and exodermis of rice roots in response to the supply of ammonium ions

The mRNA and protein for NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in root tips of rice (Oryza sativa L. cv. Sasanishiki) plants increases dramatically within 12 h of supplying a␣low concentration (>0.05 mM) of ammonium ions (T.␣Yamaya et al., 1995, Plant Cell Physiol 36: 1197–1204). To identify the specific cells which are responsible for this rapid increase, the cellular localization of NADH-GOGAT protein was investigated immunocytologically with an affinity-purified anti-NADH-GOGAT immunoglobulin G. When root tips (>1 mm) of rice seedlings which had been grown for 26 d in water were immuno-stained, signals for the NADH-GOGAT protein were detected in the central cylinder, in the apical meristem, and in the primordia of the secondary roots. Signals for ferredoxin-dependent GOGAT (Fd-GOGAT; EC 1.4.7.1) protein were also seen in the same three areas. When the roots were supplied with 1 mM ammonium ions for 24 h, there were strong signals for the NADH-GOGAT protein in two cell layers of the root surface, i.e. epidermis and exodermis, in addition to the cells giving signals in the absence of ammonium ions. The supply of ammonium ions was less effective on the profile of signals for Fd-GOGAT. Although the supply of ammonium ions had less effect on the expression of cytosolic glutamine synthetase (GS; EC 6.3.1.2), this enzyme was also found to be located in the epidermis and exodermis, as well as in the central cylinder and cortex. The results indicate that NADH-GOGAT, coupled to the cytosolic GS reaction, is probably important for the assimilation of ammonium ions in the two cell layers of the root surface. Received: 21 June 1997 / Accepted: 11 September 1997     

Tyrosine Phosphorylation and Src Family Kinases Control Keratinocyte Cell–Cell Adhesion

In their progression from the basal to upper differentiated layers of the epidermis, keratinocytes undergo significant structural changes, including establishment of close intercellular contacts. An important but so far unexplored question is how these early structural events are related to the biochemical pathways that trigger differentiation. We show here that β-catenin, γ-catenin/plakoglobin, and p120-Cas are all significantly tyrosine phosphorylated in primary mouse keratinocytes induced to differentiate by calcium, with a time course similar to that of cell junction formation. Together with these changes, there is an increased association of α-catenin and p120-Cas with E-cadherin, which is prevented by tyrosine kinase inhibition. Treatment of E-cadherin complexes with tyrosine-specific phosphatase reveals that the strength of α-catenin association is directly dependent on tyrosine phosphorylation. In parallel with the biochemical effects, tyrosine kinase inhibition suppresses formation of cell adhesive structures, and causes a significant reduction in adhesive strength of differentiating keratinocytes. The Fyn tyrosine kinase colocalizes with E-cadherin at the cell membrane in calcium-treated keratinocytes. Consistent with an involvement of this kinase, fyn-deficient keratinocytes have strongly decreased tyrosine phosphorylation levels of β- and γ-catenins and p120-Cas, and structural and functional abnormalities in cell adhesion similar to those caused by tyrosine kinase inhibitors. Whereas skin of fyn−/− mice appears normal, skin of mice with a disruption in both the fyn and src genes shows intrinsically reduced tyrosine phosphorylation of β-catenin, strongly decreased p120-Cas levels, and important structural changes consistent with impaired keratinocyte cell adhesion. Thus, unlike what has been proposed for oncogene-transformed or mitogenically stimulated cells, in differentiating keratinocytes tyrosine phosphorylation plays a positive role in control of cell adhesion, and this regulatory function appears to be important both in vitro and in vivo.     

Inflammatory cytokine‐mediated induction of serine racemase in atopic dermatitis

Serine racemase (SR) is an enzyme that catalyses the synthesis of d ‐serine, an endogenous coagonist for N‐methyl‐D‐aspartate (NMDA)‐type glutamate receptor in the central nervous system. Our previous study demonstrated that SR was expressed in the epidermis of wild‐type (WT) mice but not in SR knockout (KO) mice. In addition, SR immune‐reactivity was only found in the granular and cornified layers of the epidermis in WT mice. These findings suggested that SR is involved in the differentiation of epidermal keratinocytes and the formation of the skin barrier. However, its role in skin barrier dysfunction such as atopic dermatitis (AD) remains elusive. AD is a chronic inflammatory disease of skin, and the clinical presentation of AD has been reported to be occasionally associated with psychological factors. Therefore, this study examined the content of d ‐serine in stratum corneum in AD patients and healthy controls using a tape‐stripping method. Skin samples were collected from the cheek and upper arm skin of AD patient's lesion and healthy individuals. The d ‐serine content was significantly increased in the involved skin of AD in comparison with healthy individuals. An immunohistochemical analysis also revealed an increased SR expression in the epidermis of AD patients. Furthermore, the SR expression in cultured human keratinocytes was significantly increased by the stimulation with tumour necrosis factor ‐α or macrophage migration inhibitory factor. Taken together, these findings suggest that d ‐serine expressed particularly strongly in AD lesional skin and that the SR expression in the keratinocytes is linked to inflammatory cytokines.     

Kinetic properties and ammonium-dependent regulation of cytosolic isoenzymes of glutamine synthetase in Arabidopsis

Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme of nitrogen assimilation, catalyzing the synthesis of glutamine from ammonium and glutamate. In Arabidopsis, cytosolic GS (GS1) was accumulated in roots when plants were excessively supplied with ammonium; however, the GS activity was controlled at a constant level. The discrepancy between the protein content and enzyme activity of GS1 was attributable to the kinetic properties and expression of four distinct isoenzymes encoded by GLN1;1, GLN1;2, GLN1;3 and GLN1;4, genes that function complementary to each other in Arabidopsis roots. GLN1;2 was the only isoenzyme significantly up-regulated by ammonium, which correlated with the rapid increase in total GS1 protein. GLN1;2 was localized in the vasculature and exhibited low affinities to ammonium (Km = 2450 +/- 150 microm) and glutamate (Km = 3.8 +/- 0.2 mm). The expression of the counterpart vascular tissue-localizing low affinity isoenzyme, GLN1;3, was not stimulated by ammonium; however, the enzyme activity of GLN1;3 was significantly inhibited by a high concentration of glutamate. By contrast, the high affinity isoenzyme, GLN1;1 (Km for ammonium < 10 microm; Km for glutamate = 1.1 +/- 0.4 mm) was abundantly accumulated in the surface layers of roots during nitrogen limitation and was down-regulated by ammonium excess. GLN1;4 was another high affinity-type GS1 expressed in nitrogen-starved plants but was 10-fold less abundant than GLN1;1. These results suggested that dynamic regulations of high and low affinity GS1 isoenzymes at the levels of mRNA and enzyme activities are dependent on nitrogen availabilities and may contribute to the homeostatic control of glutamine synthesis in Arabidopsis roots.     

Taurine levels and localisation in the stratified squamous epithelia

The content and distribution of the amino acid taurine in squamous epithelia were studied using high-performance liquid chromatography and immunohistochemical methods. Quantitative analysis demonstrated that taurine was highly concentrated in the epidermis (5.49 mumol/g fresh tissue in the hairless skin of the hind footpad of the rat), although the values in the isolated stratum corneum were extremely low (< 0.073 mumol/g in the horny layer of the same skin area). No other analysed amino acid (such as glutamate, glutamine, glycine or alanine) showed this specific pattern of distribution. The immunohistochemical study revealed that in the dog and rat epidermis, taurine was present in the keratinocytes of the granular and upper spinous layers. The basal layer, lower spinous layer and stratum corneum were immunonegative. A similar immunostaining pattern was found in the epithelia of the different organs studied: the mouth, tongue and oesophagus of the dog and rat, the rat forestomach and the rat corneal epithelium. Other cell types, such as sebaceous and muscle cells, were immunolabelled. The existence of a circulating pool of taurine in the epidermis (via taurine release from keratinocytes before they reach the horny layer and its uptake by nearby cells) and its possible roles in these cells are discussed.     

AKT-dependent HspB1 (Hsp27) activity in epidermal differentiation

AKT activity has been reported in the epidermis associated with keratinocyte survival and differentiation. We show in developing skin that Akt activity associates first with post-proliferative, para-basal keratinocytes and later with terminally differentiated keratinocytes that are forming the fetal stratum corneum. In adult epidermis the dominant Akt activity is in these highly differentiated granular keratinocytes, involved in stratum corneum assembly. Stratum corneum is crucial for protective barrier activity, and its formation involves complex and poorly understood processes such as nuclear dissolution, keratin filament aggregation, and assembly of a multiprotein cell cornified envelope. A key protein in these processes is filaggrin. We show that one target of Akt in granular keratinocytes is HspB1 (heat shock protein 27). Loss of epidermal HspB1 caused hyperkeratinization and misprocessing of filaggrin. Akt-mediated HspB1 phosphorylation promotes a transient interaction with filaggrin and intracellular redistribution of HspB1. This is the first demonstration of a specific interaction between HspB1 and a stratum corneum protein and indicates that HspB1 has chaperone activity during stratum corneum formation. This work demonstrates a new role for Akt in epidermis.     

Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and α2β1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface

Single cell suspensions of human keratinocytes when seeded onto floating three-dimensional gels constructed with type I collagen form a tissue resembling epidermis. These morphogenetic events occur in a serum-free environment in the absence of fibroblasts. Light and transmission electron microscopy show that cells form a basal layer plus suprabasilar cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasilar keratinocyte layers show morphologies which resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-fillagrin granules. The basal cell layer differs from skin in vivo in that there is no connection to a basement membrane via hemidesmosomes. Cells in the basal layers are polarized as evidenced by the secretion of type IV collagen, heparan sulfate proteoglycans, and laminin at the cell membrane interface with the collagen gel. These proteins are not organized into a cytological basement membrane. Bullous pemphigoid antigen, a protein component of hemidesmosomes, is synthesized by basal keratinocytes, but like the basement membrane proteins it is not incorporated into a definable cytological structure. Keratinocytes in the basal and suprabasilar layers also synthesize α2β1 integrins. The mechanisms of keratinocyte adhesion to the gel may be through the interactions of this cell surface receptor with laminin and type IV collagen synthesized by the cell and/or direct interactions between the receptor and type I collagen within the gel. This in vitro experimental system is a useful model for defining the molecular events which control the formation and turnover of basement membranes and the mechanisms by which keratinocytes adhere to type I collagen when sheets of keratinocytes are used clinically for wound coverage.     

Relationships between nitrogenase,glutamine synthetase,glutamine, and energy charge in Azotobacter vinelandii

When continuous cultures of Azotobacter vinelandii were supplied with ammonium or nitrate in amounts, which just repressed nitrogenase synthesis completely, both the intracellular glutamine level and the degree of adenylylation of the glutamine synthetase (GS) increased only slightly (from 0.45–0.50 mM and from 2 to 3 respectively), while the total GS level remained unaffected. Higher amounts of ammonium additionally inhibited the nitrogenase activity, caused a strong rise in the intracellular glutamine concentration and adenylylation of the GS, but caused no change in the ATP/ADP ratio. These results are considered as evidence that in A. vinelandii the regulation of nitrogenase synthesis is not linked to the adenylylation state of the GS and to the intracellular glutamine level, and that the inhibition of the nitrogenase activity as a consequence of a high extracellular ammonium level is not mediated via a change in the energy charge.Abbreviations GS glutamine synthetase - GS-S(Mg) Mg²⁺ dependent synthetic activity of GS - GS-T(Mn) Mn²⁺ dependent transferase activity of GS     

Kinetic flux profiling dissects nitrogen utilization pathways in the oleaginous green alga Chlorella protothecoides

As a promising candidate for biodiesel production, the green alga Chlorella protothecoides can efficiently produce oleaginous biomass and the lipid biosynthesis is greatly influenced by the availability of nitrogen source and corresponding nitrogen assimilation pathways. Based on isotope‐assisted kinetic flux profiling (KFP), the fluxes through the nitrogen utilization pathway were quantitatively analyzed. We found that autotrophic C. protothecoides cells absorbed ammonium mainly through glutamate dehydrogenase (GDH), and partially through glutamine synthetase (GS), which was the rate‐limiting enzyme of nitrogen assimilation process with rare metabolic activity of glutamine oxoglutarate aminotransferase (GOGAT, also known as glutamate synthase); whereas under heterotrophic conditions, the cells adapted to GS‐GOGAT cycle for nitrogen assimilation in which GS reaction rate was associated with GOGAT activity. The fact that C. protothecoides chooses the adenosine triphosphate‐free and less ammonium‐affinity GDH pathway, or alternatively the energy‐consuming GS‐GOGAT cycle with high ammonium affinity for nitrogen assimilation, highlights the metabolic adaptability of C. protothecoides exposed to altered nitrogen conditions.     

Impaired Synthesis of Erythropoietin,Glutamine Synthetase and Metallothionein in the Skin of NOD/SCID/γ<Stack><Subscript>c</Subscript><Superscript>null</Superscript></Stack> and Foxn1 nu/nu Mice with Misbalanced Production of MHC Class II Complex

Most skin pathologies are characterized by unbalanced synthesis of major histocompatability complex II (MHC-II) proteins. Healthy skin keratinocytes simultaneously produce large amounts of MHC-II and regeneration-supporting proteins, e.g. erythropoietin (EPO), EPO receptor (EPOR), glutamine synthetase (GS) and metallothionein (MT). To investigate the level of regeneration-supporting proteins in the skin during misbalanced production of MHC-II, skin sections from nonobese diabetic/severe combined immunodeficient (NOD/SCID)/γ_c^null and or Foxn1 nu/nu mice which are a priory known to under- and over-express MHC II, respectively, were used. Double immunofluorescence analysis of NOD/SCID/γ_c^null skin sections showed striking decrease in expression of MHC-II, EPO, GS and MT. In Foxn1 nu/nu mouse skin, GS was strongly expressed in epidermis and in hair follicles (HF), which lacked EPO. In nude mouse skin EPO and MHC-II were over-expressed in dermal fibroblasts and they were completely absent from cortex, channel, medulla and keratinocytes surrounding the HF, suggest a role for EPO in health and pathology of hair follicle. The level of expression of EPO and GS in both mutant mice was confirmed by results of Western blot analyses. Strong immunoresponsiveness of EPOR in the hair channels of NOD/SCID/γ_c^null mouse skin suggests increased requirements of skin cells for EPO and possible benefits of exogenous EPO application during disorders of immune system accompanied by loss MHC-II in skin cells.     

Vitamin D regulated keratinocyte differentiation

The epidermis is the largest organ in the body. It is comprised primarily of keratinocytes which are arranged in layers that recapitulates their programmed life cycle. Proliferating keratinocytes are on the bottom-the stratum basale. As keratinocytes leave the stratum basale they begin to differentiate, culminating in the enucleated stratum corneum which has the major role of permeability barrier. Calcium and the active metabolite of vitamin D, 1,25(OH)(2)D(3), play important roles in this differentiation process. The epidermis has a gradient of calcium with lowest concentrations in the stratum basale, and highest concentrations in the stratum granulosum where proteins critical for barrier function are produced. Vitamin D is made in different layers of the epidermis, but 1,25(OH)(2)D(3) is made primarily in the stratum basale. Together calcium and 1,25(OH)(2)D(3) regulate the ordered differentiation process by the sequential turning on and off the genes producing the elements required for differentiation as well as activating those enzymes involved in differentiation. Animal models in which the sensing mechanism for calcium, the receptor for 1,25(OH)(2)D(3), or the enzyme producing 1,25(OH)(2)D(3) have been rendered inoperative demonstrate the importance of these mechanisms for the differentiation process, although each animal model has its own phenotype. This review will examine the mechanisms by which calcium and 1,25(OH)(2)D(3) interact to control epidermal differentiation.     

Expression of acyl-CoA synthetase 5 in human epidermis

The human epidermis is characterized by a constant renewal of keratinocytes embedded in a matrix enriched with lipids. Numerous proteins involved in lipid metabolism are found in human epidermis, especially in keratinocytes. Long-chain acyl-CoA derivatives, which are catalyzed by human ACSL5, are important metabolites in several biochemical pathways, including ceramide de novo synthesis. The aim of the present study was to investigate expression of acyl-CoA synthetase isoform 5 (ACSL5) in human epidermis by an in situ, as well as a molecular approach. We show that ACSL5 mRNA and protein are found in human epidermis, as well as in non-differentiated and differentiated HaCaT cells. Keratinocytes of stratum spinosum are the main source for ACSL5 expression in both meshed facial or abdominal skin and ridged skin of upper or lower extremities including TUNEL-positive cells in upper cellular layers. Single keratinocytes of chronic solar-exposed meshed facial epidermis occasionally display a stronger ACSL5 immunostaining. In conclusion, our study indicates that epidermal ACSL5 expression might be involved in differentiation and the stress response of keratinocytes.     

p120ctn and P-Cadherin but Not E-Cadherin Regulate Cell Motility and Invasion of DU145 Prostate Cancer Cells

Background

Adherens junctions consist of transmembrane cadherins, which interact intracellularly with p120ctn, ß-catenin and α-catenin. p120ctn is known to regulate cell-cell adhesion by increasing cadherin stability, but the effects of other adherens junction components on cell-cell adhesion have not been compared with that of p120ctn.

Methodology/Principal Findings

We show that depletion of p120ctn by small interfering RNA (siRNA) in DU145 prostate cancer and MCF10A breast epithelial cells reduces the expression levels of the adherens junction proteins, E-cadherin, P-cadherin, ß-catenin and α-catenin, and induces loss of cell-cell adhesion. p120ctn-depleted cells also have increased migration speed and invasion, which correlates with increased Rap1 but not Rac1 or RhoA activity. Downregulation of P-cadherin, β-catenin and α-catenin but not E-cadherin induces a loss of cell-cell adhesion, increased migration and enhanced invasion similar to p120ctn depletion. However, only p120ctn depletion leads to a decrease in the levels of other adherens junction proteins.

Conclusions/Significance

Our data indicate that P-cadherin but not E-cadherin is important for maintaining adherens junctions in DU145 and MCF10A cells, and that depletion of any of the cadherin-associated proteins, p120ctn, ß-catenin or α-catenin, is sufficient to disrupt adherens junctions in DU145 cells and increase migration and cancer cell invasion.