scholarly journals Phosphatidylinositol-4-phosphate 5-kinase 1α Mediates Extracellular Calcium-induced Keratinocyte Differentiation

2009 ◽  
Vol 20 (6) ◽  
pp. 1695-1704 ◽  
Author(s):  
Zhongjian Xie ◽  
Sandra M. Chang ◽  
Sally D. Pennypacker ◽  
Er-Yuan Liao ◽  
Daniel D. Bikle
Keyword(s):  
Plasma Membrane ◽  
Second Messengers ◽  
Human Keratinocytes ◽  
Extracellular Calcium ◽  
Keratinocyte Differentiation ◽  
Activity Levels ◽  
Differentiation Markers ◽  
Resultant Increase ◽  
Phosphatidylinositol 4 ◽  
E Cadherin

Extracellular calcium (Cao) is a major regulator of keratinocyte differentiation, but the mechanism is unclear. Phosphatidylinositol-4-phosphate 5-kinase 1α (PIP5K1α) is critical in synthesizing phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. In this study, we sought to determine whether PIP5K1α plays a role in mediating the ability of Cao to induce keratinocyte differentiation. We found that treatment of human keratinocytes in culture with Cao resulted in increased PIP5K1α level and activity, as well as PI(4,5)P2 level, binding of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] to and activation of phospholipase C-γ1 (PLC-γ1), with the resultant increase in inositol 1,4,5-trisphosphate (IP3) and intracellular calcium (Cai). Knockdown of PIP5K1α in human keratinocytes blocked Cao-induced increases in the binding of PI(3,4,5)P3 to PLC-γ1; PLC-γ1 activity; levels of PI(4,5)P2, IP3, and Cai; and induction of keratinocyte differentiation markers. Coimmunoprecipitation and confocal studies revealed that Cao stimulated PIP5K1α recruitment to the E-cadherin–catenin complex in the plasma membrane. Knockdown of E-cadherin or β-catenin blocked Cao-induced activation of PIP5K1α. These results indicate that after Cao stimulation PIP5K1α is recruited by the E-cadherin–catenin complex to the plasma membrane where it provides the substrate PI(4,5)P2 for both PI3K and PLC-γ1. This signaling pathway is critical for Cao-induced generation of the second messengers IP3 and Cai and keratinocyte differentiation.

scholarly journals Phosphoprotein Phosphatase 1 Is Required for Extracellular Calcium-Induced Keratinocyte Differentiation

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Chandrama Shrestha ◽  
Yuanyuan Tang ◽  
Hong Fan ◽  
Lusha Li ◽  
Qin Zeng ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Second Messengers ◽  
Human Keratinocytes ◽  
Extracellular Calcium ◽  
Keratinocyte Differentiation ◽  
Differentiation Markers ◽  
Calcium Stimulation ◽  
E Cadherin

Extracellular calcium is a major regulator of keratinocyte differentiation in vitro and appears to play that role in vivo, but the mechanism is unclear. We have previously demonstrated that, following calcium stimulation, PIP5K1αis recruited by the E-cadherin-β-catenin complex to the plasma membrane where it provides the substrate PIP2 for both PI3K and PLC-γ1. This signaling pathway is critical for calcium-induced generation of second messengers including IP3 and intracellular calcium and keratinocyte differentiation. In this study, we explored the upstream regulatory mechanism by which calcium activates PIP5K1αand the role of this activation in calcium-induced keratinocyte differentiation. We found that treatment of human keratinocytes in culture with calcium resulted in an increase in serine dephosphorylation and PIP5K1αactivation. PP1 knockdown blocked extracellular calcium-induced increase in serine dephosphorylation and activity of PIP5K1αand induction of keratinocyte differentiation markers. Knockdown of PLC-γ1, the downstream effector of PIP5K1α, blocked upstream dephosphorylation and PIP5K1αactivation induced by calcium. Coimmunoprecipitation revealed calcium induced recruitment of PP1 to the E-cadherin-catenin-PIP5K1αcomplex in the plasma membrane. These results indicate that PP1 is recruited to the extracellular calcium-dependent E-cadherin-catenin-PIP5K1αcomplex in the plasma membrane to activate PIP5K1α, which is required for PLC-γ1 activation leading to keratinocyte differentiation.

Inhibition of cadherin function differentially affects markers of terminal differentiation in cultured human keratinocytes

1999 ◽  
Vol 112 (24) ◽  
pp. 4569-4579
Author(s):  
M.D. Hines ◽  
H.C. Jin ◽  
M.J. Wheelock ◽  
P.J. Jensen
Keyword(s):  
Terminal Differentiation ◽  
Human Keratinocytes ◽  
Keratinocyte Differentiation ◽  
Differentiation Markers ◽  
Protein Levels ◽  
Surface Molecules ◽  
Transglutaminase 1 ◽  
Differentiation Marker ◽  
E Cadherin

Cadherin function is required for normal keratinocyte intercellular adhesion and stratification. In the present study, we have investigated whether cadherin-cadherin interactions may also modulate keratinocyte differentiation, as evidenced by alterations in the levels of several differentiation markers. Confluent keratinocyte cultures, propagated in low Ca(2+) medium in which cadherins are not active, were pre-incubated with antibodies that block the function of E-cadherin and/or P-cadherin; Ca(2+)was then elevated to 1 mM to activate the cadherins and induce differentiation. In control cultures (incubated with no antibody or with antibodies to other cell surface molecules), Ca(2+) elevation induced an increase in type 1 transglutaminase, profilaggrin, and loricrin, as measured by western blotting and in agreement with previous results. However, the concurrent addition of antibodies against both E- and P-cadherin prevented this increase in transglutaminase 1 protein. Incubation with either antibody alone had no consistent effect. Profilaggrin and loricrin, which are later markers of keratinocyte differentiation, responded differently from transglutaminase 1 to addition of antibodies. In the presence of anti-E-cadherin antibody, both loricrin and profilaggrin levels were dramatically enhanced compared to the high Ca(2+) control cells, while addition of antibody to P-cadherin slightly attenuated the Ca(2+)-induced increase. In the presence of both antibodies, loricrin and profilaggrin protein levels were intermediate between those observed in the presence of either antibody alone. The expression of involucrin, however, was unaffected by addition of antibodies. In addition, effects of the anti-cadherin antibodies were not secondary to alterations in proliferation or programmed cell death, as determined by several independent assays of these processes. Thus, the consequences of cadherin inhibition depend upon both the particular cadherin and the differentiation marker under study. Taken together, these data suggest that E-cadherin and P-cadherin contribute to the orderly progression of terminal differentiation in the epidermis in multiple ways.

scholarly journals Addressing differentiation in live human keratinocytes by assessment of membrane packing order

2020 ◽  
Author(s):  
Danuta Gutowska-Owsiak ◽  
Christian Eggeling ◽  
Graham S Ogg ◽  
Jorge Bernardino de la Serna
Keyword(s):  
Stratum Corneum ◽  
Single Cell ◽  
Human Keratinocytes ◽  
Keratinocyte Differentiation ◽  
Dead Cell ◽  
Differentiation Markers ◽  
Complex Processes ◽  
Impermeable Barrier ◽  
Therapeutic Development ◽  
Polarity Sensitive

Differentiation of keratinocytes is critical for epidermal stratification and formation of a protective stratum corneum. It involves a series of complex processes leading through gradual changes in characteristics and functions of keratinocytes up to their programmed cell death via cornification. The stratum corneum is an impermeable barrier, comprised of dead cell remnants (corneocytes) embedded within lipid matrix. Corneocyte membranes are comprised of specialized lipids linked to late differentiation proteins, contributing to the formation of a highly stiff and mechanically strengthen layer. To date, the assessment of the progression of keratinocyte differentiation is only possible by determination of specific differentiation markers, e.g. by using proteomics-based approaches. Unfortunately, this requires fixation or cell lysis, and currently there is no robust methodology available to study differentiation in living cells, neither at a single cell, nor in high throughput. Here, we explore a new live-cell based approaches for screening differentiation advancement in keratinocytes, in a “calcium switch” model. We employ a polarity-sensitive dye, Laurdan, and Laurdan general polarization function (GP) as a reporter of the degree of membrane lateral packing order or condensation, as an adequate marker of differentiation. We show that the assay is straightforward and can be conducted either on a single cell level using confocal spectral imaging or on the ensemble level using a fluorescence plate reader. Such systematic quantification may become useful for understanding mechanisms of keratinocyte differentiation, such as the role of membrane inhomogeneities in stiffness, and for future therapeutic development.

scholarly journals 2-APB arrests human keratinocyte proliferation and inhibits cutaneous squamous cell carcinomain vitro

2018 ◽  
Author(s):  
Aislyn M. Nelson ◽  
Yalda Moayedi ◽  
Sophie A. Greenberg ◽  
Marlon E. Ruiz ◽  
Uffe B. Jensen ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Risk ◽  
Cell Lines ◽  
Squamous Cell ◽  
Transient Receptor Potential ◽  
Human Keratinocytes ◽  
Keratinocyte Differentiation ◽  
Differentiation Markers ◽  
Keratinocyte Proliferation ◽  
Human Keratinocyte

AbstractBackgroundThe epidermis is a stratified epithelium whose differentiation program is triggered in part by calcium. Dysregulation of keratinocyte differentiation may lead to non-melanoma skin cancers, including cutaneous squamous cell carcinoma (cSCC). The compound 2-aminoethoxydiphenyl borate (2-APB) modulates calcium signaling by altering activity of calcium-permeable channels of the transient receptor potential (TRP) and ORAI families, and is therefore poised to govern signaling pathways that control the balance of keratinocyte proliferation and differentiation.ObjectiveWe sought to determine whether 2-APB alters differentiation of normal human keratinocytes and progression of human cSCCs modelsin vitro.MethodsPrimary human keratinocyte cultures were treated with 2-APB and levels of proliferation (EdU incorporation) and differentiation markers [quantitative PCR (qPCR)] were assessed. Human cSCC biopsies and cell lines were analyzed for TRP and ORAI gene expression via qPCR. cSCC cell lines were cultured in organtypic cultures and analyzed for growth and invasiveness after 2-APB or vehicle treatment.ResultsCulturing human keratinocytes with 2-APB arrested cell proliferation, triggered differentiation-gene expression and altered epidermal stratification, indicating that 2-APB application is sufficient to promote differentiation. In human organotypic cSCC cultures, 2-APB attenuated tumor growth and invasiveness. Finally, expression of a panel of 2-APB-targeted ion channels (TRPV3, TRPV1, TRPC1, OraI1, OraI2 and OraI3) was dysregulated in high-risk cSCC biopsies.ConclusionsCollectively, these findings identify 2-APB as a potential therapeutic for high-risk cSCCs.

scholarly journals Commitment to differentiation and expression of early differentiation markers in murine keratinocytes in vitro are regulated independently of extracellular calcium concentrations.

1993 ◽  
Vol 123 (4) ◽  
pp. 909-919 ◽  
Author(s):  
V Drozdoff ◽  
W J Pledger
Keyword(s):  
Structural Changes ◽  
Solid Medium ◽  
Extracellular Calcium ◽  
Keratinocyte Differentiation ◽  
Differentiation Markers ◽  
Primary Mouse ◽  
Semi Solid ◽  
Mouse Keratinocytes

In the epidermis, one of the earliest characterized events in keratinocyte differentiation is the coordinate induction of a pair of keratins specifically expressed in suprabasal cells, keratin 1 (K1) and keratin 10 (K10). Both in vivo and in vitro, extracellular calcium is necessary for several biochemical and structural changes during keratinocyte differentiation. However, it has been unclear if calcium serves as a differentiation signal in keratinocytes. In these studies, expression of suprabasal keratin mRNA and protein is used to test whether the initial differentiation of primary mouse keratinocytes in vitro is dependent on changes in the concentration of extracellular calcium. K1 mRNA was expressed at low levels in cultures of keratinocytes growing on plastic in 0.05 mM calcium but in attached cells was not further induced by increases in the concentration of extracellular calcium. Suspension of the keratinocytes into semi-solid medium induced a rapid and substantial increase in both expression of K1 mRNA and in the percentage of cells expressing suprabasal keratin proteins. The induction was unaffected by the concentration of calcium in the semi-solid medium and could not be enhanced by exposing attached cells to higher calcium before suspension. The induction of K1 mRNA could be inhibited by exposure of the keratinocytes to either EGF or fibronectin. These results suggest that commitment of mouse keratinocytes to terminal differentiation is independent of extracellular calcium and may be regulated primarily by extracellular factors other than calcium.

scholarly journals An association between type Iγ PI4P 5-kinase and Exo70 directs E-cadherin clustering and epithelial polarization

2012 ◽  
Vol 23 (1) ◽  
pp. 87-98 ◽  
Author(s):  
Xunhao Xiong ◽  
Qingwen Xu ◽  
Yan Huang ◽  
Raman Deep Singh ◽  
Richard Anderson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Adherens Junctions ◽  
Direct Interaction ◽  
Phosphatidylinositol 4 ◽  
E Cadherin

E-Cadherin–mediated formation of adherens junctions (AJs) is essential for the morphogenesis of epithelial cells. However, the mechanisms underlying E-cadherin clustering and AJ maturation are not fully understood. Here we report that type Iγ phosphatidylinositol-4-phosphate 5-kinase (PIPKIγ) associates with the exocyst via a direct interaction with Exo70, the exocyst subunit that guides the polarized targeting of exocyst to the plasma membrane. By means of this interaction, PIPKIγ mediates the association between E-cadherin and Exo70 and determines the targeting of Exo70 to AJs. Further investigation revealed that Exo70 is necessary for clustering of E-cadherin on the plasma membrane and extension of nascent E-cadherin adhesions, which are critical for the maturation of cohesive AJs. In addition, we observed phosphatidylinositol-4,5-bisphosphate (PI4,5P2) accumulation at E-cadherin clusters during the assembly of E-cadherin adhesions. PIPKIγ-generated PI4,5P2 is required for recruiting Exo70 to newly formed E-cadherin junctions and facilitates the assembly and maturation of AJs. These results support a model in which PIPKIγ and PIPKIγ-generated PI4,5P2 pools at nascent E-cadherin contacts cue Exo70 targeting and orient the tethering of exocyst-associated E-cadherin. This could be an important mechanism that regulates E-cadherin clustering and AJ maturation, which is essential for the establishment of solid, polarized epithelial structures.

Extracellular Calcium and cAMP: Second Messengers as “Third Messengers”?

Physiology ◽  
2007 ◽  
Vol 22 (5) ◽  
pp. 320-327 ◽  
Author(s):  
Aldebaran M. Hofer ◽  
Konstantinos Lefkimmiatis
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Cell Surface ◽  
Second Messengers ◽  
Signaling Molecules ◽  
Extracellular Calcium ◽  
Cell Surface Receptors ◽  
Surface Receptors

Calcium and cyclic AMP are familiar second messengers that typically become elevated inside cells on activation of cell surface receptors. This article will explore emerging evidence that transport of these signaling molecules across the plasma membrane allows them to be recycled as “third messengers,” extending their ability to convey information in a domain outside the cell.

scholarly journals Glycosyl-phosphatidylinositol-anchored membrane proteins.

1992 ◽  
Vol 3 (4) ◽  
pp. 895-906 ◽  
Author(s):  
D Brown ◽  
G L Waneck
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Transmembrane Domain ◽  
Second Messengers ◽  
Differentiation Markers ◽  
Coated Pits ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Type Iv ◽  
Activation Antigens

Many proteins of eukaryotic cells are anchored to membranes by covalent linkage to glycosyl-phosphatidylinositol (GPI). These proteins lack a transmembrane domain, have no cytoplasmic tail, and are, therefore, located exclusively on the extracellular side of the plasma membrane. GPI-anchored proteins form a diverse family of molecules that includes membrane-associated enzymes, adhesion molecules, activation antigens, differentiation markers, protozoan coat components, and other miscellaneous glycoproteins. In the kidney, several GPI-anchored proteins have been identified, including uromodulin (Tamm-Horsfall glycoprotein), carbonic anhydrase type IV, alkaline phosphatase, Thy-1, BP-3, aminopeptidase P, and dipeptidylpeptidase. GPI-anchored proteins can be released from membranes with specific phospholipases and can be recovered from the detergent-insoluble pellet after Triton X-114 treatment of membranes. All GPI-anchored proteins are initially synthesized with a transmembrane anchor, but after translocation across the membrane of the endoplasmic reticulum, the ecto-domain of the protein is cleaved and covalently linked to a preformed GPI anchor by a specific transamidase enzyme. Although it remains obscure why so many proteins are endowed with a GPI anchor, the presence of a GPI anchor does confer some functional characteristics to proteins: (1) it is a strong apical targeting signal in polarized epithelial cells; (2) GPI-anchored proteins do not cluster into clathrin-coated pits but instead are concentrated into specialized lipid domains in the membrane, including so-called smooth pinocytotic vesicles, or caveoli; (3) GPI-anchored proteins can act as activation antigens in the immune system; (4) when the GPI anchor is cleaved by PI-phospholipase C or PI-phospholipase D, second messengers for signal transduction may be generated; (5) the GPI anchor can modulate antigen presentation by major histocompatibility complex molecules. Finally, at least one human disease, paroxysmal nocturnal hemoglobinuria, is a result of defective GPI anchor addition to plasma membrane proteins.

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