A sensitive method to quantify the terminal differentiation of cultured epidermal cells

King, Ivan; Mella, Sharon L.; Sartorelli, Alan C.

doi:10.1016/0014-4827(86)90221-1

Cited by 38 publications

(18 citation statements)

References 9 publications

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“…(42). In this method, the cornified envelope precursor proteins were labelled with [35S]methionine by incubating cultures with [35S]-L methionine (2 MCi/ml medium) for 48 h. These precursors were cross-linked by the epidermal transglutaminase to form a detergent-insoluble, thickened envelope which surrounds the terminally differentiated keratinocytes.…”

Section: Methodsmentioning

confidence: 99%

Adenosine triphosphate stimulates phosphoinositide metabolism, mobilizes intracellular calcium, and inhibits terminal differentiation of human epidermal keratinocytes.

Pillai

Bikle²

1992

J. Clin. Invest.

108

101

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During wound healing, release of ATP from platelets potentially exposes the epidermis to concentrations of ATP known to alter cellular functions mediated via changes in inositol trisphosphate (IP3) and intracellular calcium (Cai) levels. Therefore, we determined whether keratinocytes respond to ATP with a rise in Cai and IP3 and whether such increases are accompanied by a change in their proliferation and differentiation. Changes in Cai were measured in Indo-l-loaded neonatal human foreskin keratinocytes after stimulation with extracellular ATP. Extracellular ATP evoked a transient and acute increase in Cai of keratinocytes both in the presence and in the absence of extracellular calcium. ATP also induced the phosphoinositide turnover of keratinocytes, consistent with its effect in releasing calcium from intracellular sources. ATP did not permeabilize keratinocytes, nor did it promote Ca influx into the cells. The half-maximal effect of ATP was at 10 MM, and saturation was observed at 30-100 ,M. UTPP, ITP, and ATP-yS were as effective as ATP in releasing Cai from intracellular stores and competed with ATP for their response, whereas AMP and adenosine were ineffective, suggesting the specificity of P2 purinergic receptors in mediating the ATP response in keratinocytes. Single cell measurements revealed heterogeneity in the calcium response to ATP. This heterogeneity did not appear to be due to differences in the initial Cai response but to subsequent removal of increased Cai by these cells. ATP inhibited terminal differentiation of keratinocytes as measured by I35Slmethionine incorporation into cornified envelopes and modestly stimulated incorporation of r3Hlthymidine into DNA.Chelation of Cai by bis-(o-aminophenoxy)-NNN',N'-tetraacetic acid reduced basal Cai, blocked the Cai response to ATP, inhibited the basal rate of DNA synthesis, and blocked the ATP-induced increase in DNA synthesis. We conclude that extracellular ATP may be an important physiological regulator of epidermal growth and differentiation acting via IP3 and Cai. (J. Clin. Invest. 1992. 90:42-51.)

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Section: Methodsmentioning

confidence: 99%

Adenosine triphosphate stimulates phosphoinositide metabolism, mobilizes intracellular calcium, and inhibits terminal differentiation of human epidermal keratinocytes.

Pillai

Bikle²

1992

J. Clin. Invest.

108

101

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“…A Effect ofTNF alpha on differentiation ofkeratinocytes. The formation of cornified envelopes was quantitated using the method of King et al (23). The cells in triplicate wells were exposed to 2 MACi of [35S]-methionine/ml media for 48 h. The washed cells were then dissolved in 2 ml of2% SDS, 20 mM DTT solution and the amount ofradioactivity incorporated into the cross-linked, detergent-insoluble, cornified envelope was quantitated by liquid scintillation spectroscopy after collecting the envelopes on 10-Mm pore size filters.…”

Section: Methodsmentioning

confidence: 99%

Binding and biological effects of tumor necrosis factor alpha on cultured human neonatal foreskin keratinocytes.

Pillai

Bikle²,

Eessalu³

et al. 1989

J. Clin. Invest.

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Tumor necrosis factor alpha (TNF alpha) localizes to the epidermis when injected in vivo, but its role in the skin has heretofore not been evaluated. As a first approach to assessing the role of TNF alpha in the skin, we evaluated the binding and biological effects of TNF alpha on human neonatal foreskin keratinocytes maintained in culture. We found that TNF alpha at 0.3-1.0 nM inhibited proliferation of keratinocytes in a reversible fashion as demonstrated by a reduction in total DNA content and clonal growth. The antiproliferative effects were most marked when TNF alpha was added in the preconfluent stages of cell growth. Accompanying this antiproliferative effect was a stimulation by TNF alpha of differentiation of keratinocytes as indicated by the stimulation of cornified envelope formation. Keratinocytes specifically bound TNF alpha, reaching maximal binding in 2 h at 340C or 8 h at 4VC. Much of the apparent binding at 340C was due to internalization of the TNF alpha. At 4VC the rate of internalization was much less. Confluent keratinocytes showed a single class of high-affinity receptors with 1,250 receptors/cell and a Kd of 0.28 nM. These data suggest a role for TNF alpha in the growth and differentiation of the epidermis.

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“…Relative levels of cornified envelopes in HaCaT cell cultures were determined by the radiolabel method of King et al [16]. Briefly, the cells were labeled with [ 3 H]lysine for 48 h and were then harvested by scraping them into the medium and centrifuging them at 1,500 g for 10 min.…”

Section: Cornificationmentioning

confidence: 99%

The Induction of Terminal Differentiation Markers by the cAMP Pathway in Human HaCaT Keratinocytes

Mammone

Marenus

Maes

et al. 1998

Skin Pharmacol Physiol

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The terminal differentiation of human epidermal keratinocytes is a complex morphological and biochemical shift from a mitotically active cell to an inert protein cross-linked envelope. This transition is a clearly predetermined cell death mechanism, but it is unlike many other programmed cell deaths in that it is not apoptotic. To explore and contrast the mechanism by which keratinocytes are committed to differentiation rather than apoptosis, we focused on the cyclic adenosine monophosphate (cAMP) signaling pathway using selective modulators of intracelluar cAMP levels. Markers of differentiation were assayed by Western blotting. Raising intracelluar cAMP levels by treating HaCaT cells with forskolin, a diterpene, or with isobutylmethylxanthine, a phosphodiesterase inhibitor, and isoproterenol, a β-adrenergic receptor agonist that selectively activates adenylate cyclase, increased the levels of the differentiation markers keratin K1 and K10, involucrin and transglutaminase. H89 and KT5720, both inhibitors of cAMP-dependent protein kinase, suppressed the expression of keratins K1 and K10. These observations are in line with the defined role for cAMP in the control of keratinocyte differentiation.

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A sensitive method to quantify the terminal differentiation of cultured epidermal cells

Cited by 38 publications

References 9 publications

Adenosine triphosphate stimulates phosphoinositide metabolism, mobilizes intracellular calcium, and inhibits terminal differentiation of human epidermal keratinocytes.

Adenosine triphosphate stimulates phosphoinositide metabolism, mobilizes intracellular calcium, and inhibits terminal differentiation of human epidermal keratinocytes.

Binding and biological effects of tumor necrosis factor alpha on cultured human neonatal foreskin keratinocytes.

The Induction of Terminal Differentiation Markers by the cAMP Pathway in Human HaCaT Keratinocytes

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