Formation of the Epidermal Calcium Gradient Coincides with Key Milestones of Barrier Ontogenesis in the Rodent
1998; Elsevier BV; Volume: 110; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1747.1998.00151.x
ISSN1523-1747
AutoresPeter M. Elias, P. Nau, Karen Piper Hanley, Chris Cullander, Debra Crumrine, Graham Bench, E. Sideras‐Haddad, Theodora M. Mauro, Mary L. Williams, Kenneth R. Feingold,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoThe epidemal permeability barrier forms late in gestation, coincident with decreased lipid synthesis, increased lipid processing, and development of a mature, multilayered stratum corneum. Prior studies have shown that changes in the epidermal Ca++ gradient in vivo regulate lamellar body secretion and lipid synthesis, and modulations in extracellular Ca++in vitro also regulate keratinocyte differentiation. We asked here whether a Ca++ gradient forms in fetal epidermis in utero, and whether its emergence correlates with key developmental milestones of barrier formation and stratum corneum development. Using either ion precipitation or proton induced X-ray emission analysis of fetal mouse and rat skin, we showed that a Ca++ gradient is not present at gestational days 16–18, prior to barrier formation, and that a gradient forms coincident with the emergence of barrier competence (day 19, mouse; day 20, rat) prior to birth. These results are consistent with a role for Ca++in the regulation of key metabolic events leading to barrier formation. Whether the calcium gradient is formed actively or passively remains to be determined. The epidemal permeability barrier forms late in gestation, coincident with decreased lipid synthesis, increased lipid processing, and development of a mature, multilayered stratum corneum. Prior studies have shown that changes in the epidermal Ca++ gradient in vivo regulate lamellar body secretion and lipid synthesis, and modulations in extracellular Ca++in vitro also regulate keratinocyte differentiation. We asked here whether a Ca++ gradient forms in fetal epidermis in utero, and whether its emergence correlates with key developmental milestones of barrier formation and stratum corneum development. Using either ion precipitation or proton induced X-ray emission analysis of fetal mouse and rat skin, we showed that a Ca++ gradient is not present at gestational days 16–18, prior to barrier formation, and that a gradient forms coincident with the emergence of barrier competence (day 19, mouse; day 20, rat) prior to birth. These results are consistent with a role for Ca++in the regulation of key metabolic events leading to barrier formation. Whether the calcium gradient is formed actively or passively remains to be determined. proton induced X-ray emission stratum corneum stratum granulosum The epidermis displays a characteristic calcium (Ca++) gradient, shown both by ion capture cytochemistry and by biophysical methods (Menon et al., 1985Menon G.K. Grayson S. Elias P.M. Ionic calcium reservoirs in mammalian epidermis: Ultrastructural localization by ion-capture cytochemistry.J Invest Dermatol. 1985; 84: 508-512Abstract Full Text PDF PubMed Scopus (390) Google Scholar; Forslind, 1987Forslind B. Quantitative X-ray microanalysis of skin.Acta Derma Vener. 1987; 134: 1-8Google Scholar). Ca++ is sparse in the basal and spinous layers, increasing to the highest levels in the granular layer (SG) of untreated epidermis, and declining again in the stratum corneum (SC). With acute barrier disruption, Ca++ levels decrease in the outer epidermis, due to displacement of this ion outward through the SC (Menon et al., 1992aMenon G.K. Elias P.M. Feingold K.R. Localization of calcium in murine epidermis following disruption and repair of the permeability barrier.Cell Tiss Res. 1992; 270: 503-512Crossref PubMed Scopus (164) Google Scholar; Mao-qiang et al., 1997Mao-qiang M. Mauro T. Bench G. Warren R. Elias P.M. Feingold K.R. Calcium and potassium inhibit barrier recovery after disruption, independent of the type of insult in hairless mice.Exp Dermatol. 1997; 6: 36-40Crossref PubMed Scopus (44) Google Scholar). The Ca++ gradient in turn is restored over 24 h in parallel with barrier recovery, but it does not return under occlusion (Menon et al., 1992aMenon G.K. Elias P.M. Feingold K.R. Localization of calcium in murine epidermis following disruption and repair of the permeability barrier.Cell Tiss Res. 1992; 270: 503-512Crossref PubMed Scopus (164) Google Scholar), just as the barrier fails to normalize under these conditions (Grubauer et al., 1989Grubauer G. Elias P.M. Feingold K.R. Transepidermal water loss. The signal for recovery of barrier structure and function.J Lipid Res. 1989; 30: 323-334Abstract Full Text PDF PubMed Google Scholar). Moreover, the Ca++ gradient is abnormal in other, more sustained or chronic forms of barrier disruption, i.e., in essential fatty acid deficiency, repeated lovastatin treatment, and psoriasis (Menon and Elias, 1991Menon G.K. Elias P.M. Ultrastructural localization of calcium in psoriatic and normal human epidermis.Arch Dennatol. 1991; 127: 57-63Crossref PubMed Scopus (169) Google Scholar; Menon et al., 1994aMenon G.K. Elias P.M. Feingold K.R. Integrity of the permeability barrier is crucial for maintenance ofthe epidermal calcium gradient.Br J Dermatol. 1994; 130: 139-147Crossref PubMed Scopus (105) Google Scholar) with increased levels of Ca++ present in all epidermal cell layers. Occlusion in these models normalizes the Ca++ gradient in parallel with partial restoration of the barrier (Menon et al., 1994aMenon G.K. Elias P.M. Feingold K.R. Integrity of the permeability barrier is crucial for maintenance ofthe epidermal calcium gradient.Br J Dermatol. 1994; 130: 139-147Crossref PubMed Scopus (105) Google Scholar). Finally, the epidermal Ca++ gradient is thought to regulate not only barrier homeostasis, but also keratinocyte/epidermal differentiation (e.g., Hennings et al., 1983Hennings H. Holbrook K.A. Yuspa S.H. Factors influencing calcium-induced terminal differentiation in cultured mouse epidermal cells.J Cellular Physiol. 1983; 116: 265-281Crossref PubMed Scopus (92) Google Scholar; Yuspa et al., 1989Yuspa S.H. Kilkenny A.K. Steinert P.M. Roop D.R. Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro.J Cell Biol. 1989; 109: 1207-1217Crossref PubMed Scopus (514) Google Scholar). Permeability barrier requirements regulate several metabolic processes in the underlying nucleated layers of the epidermis (Feingold, 1991Feingold K.R. The regulation and role of epidermal lipid synthesis.Adv Lipid Res. 1991; 24: 57-79Crossref PubMed Google Scholar; Elias, 1996Elias P.M. Stratum corneum architecture, metabolic activity, and interactivity with subjacent cell layers.Exp Dermatol. 1996; 5: 191-201Crossref PubMed Scopus (110) Google Scholar). These responses include the synthesis of all three major classes of SC lipids, the generation and secretion of epidermal lamellar bodies (LB), and epidermal DNA synthesis. The link between these responses and the barrier is demonstrated by experiments that show that artificial restoration of the barrier by occlusion with vapor-impermeable, but not vapor-permeable wraps, immediately blocks, wholly or in part, the various metabolic responses detailed above after acute barrier perturbation (Grubauer et al., 1989Grubauer G. Elias P.M. Feingold K.R. Transepidermal water loss. The signal for recovery of barrier structure and function.J Lipid Res. 1989; 30: 323-334Abstract Full Text PDF PubMed Google Scholar; Feingold, 1991Feingold K.R. The regulation and role of epidermal lipid synthesis.Adv Lipid Res. 1991; 24: 57-79Crossref PubMed Google Scholar). Yet, transepidermal water loss is not the signal for these metabolic responses, because the barrier recovers normally when perturbed skin sites are immersed in isotonic, hypertonic, or hypotonic solutions (Lee et al., 1992Lee S.H. Elias P.M. Proksch E. Menon G.K. Mao-qiang M. Feingold K.R. Calcium and potassium are important regulators of barrier homeostasis in murine epidermis.J Clin Invest. 1992; 89: 530-538Crossref PubMed Scopus (194) Google Scholar); however, when a mixture of ions, particularly Ca++ and K+, is added to the solution, recovery is blocked to an extent comparable with occlusion (Lee et al., 1992Lee S.H. Elias P.M. Proksch E. Menon G.K. Mao-qiang M. Feingold K.R. Calcium and potassium are important regulators of barrier homeostasis in murine epidermis.J Clin Invest. 1992; 89: 530-538Crossref PubMed Scopus (194) Google Scholar, Lee et al., 1994Lee S.H. Elias P.M. Feingold K.R. Mauro T. A role for ions in barrier recovery after acute perturbation.J Invest Dermatol. 1994; 102: 976-979Abstract Full Text PDF PubMed Google Scholar). Moreover, exogenous exposure to these ions also downregulates the expected increase in the activity of HMGCoA reductase, the rate limiting enzyme in cholesterol synthesis, following barrier perturbation (Lee et al., 1992Lee S.H. Elias P.M. Proksch E. Menon G.K. Mao-qiang M. Feingold K.R. Calcium and potassium are important regulators of barrier homeostasis in murine epidermis.J Clin Invest. 1992; 89: 530-538Crossref PubMed Scopus (194) Google Scholar). Ca++ accounts for many of the inhibitory effects on barrier recovery, as shown by the reversal of inhibition of barrier recovery by cotreatment with inhibitors of either L-type calcium channels or calmodulin (Lee et al., 1992Lee S.H. Elias P.M. Proksch E. Menon G.K. Mao-qiang M. Feingold K.R. Calcium and potassium are important regulators of barrier homeostasis in murine epidermis.J Clin Invest. 1992; 89: 530-538Crossref PubMed Scopus (194) Google Scholar, Lee et al., 1994Lee S.H. Elias P.M. Feingold K.R. Mauro T. A role for ions in barrier recovery after acute perturbation.J Invest Dermatol. 1994; 102: 976-979Abstract Full Text PDF PubMed Google Scholar). Finally, using high frequency sonophoresis to alter Ca++ levels in the SG, we showed that lamellar body secretion occurs in response to decreases in extracellular Ca++ , without alterations in barrier function (Menon et al., 1994bMenon G.K. Price L.F. Bommannan B. Elias P.M. Feingold K.R. Selective obliteration of the epidermal calcium gradient leads to enhanced lamellar body secretion.J Invest Dermatol. 1994; 102: 789-795Abstract Full Text PDF PubMed Google Scholar). The permeability barrier develops relatively late in fetal development. In rats, the barrier is still incompetent on day 19, with partial formation on day 20 and mature function by day 21 (gestation is on day 22) (Aszterbaum et al., 1992Aszterbaum M. Menon G.K. Feingold K.R. Williams M.L. Ontogeny of the epidermal barrier to water loss in the rat: correlation of function with stratum corneum structure and lipid content.Pediatr Res. 1992; 31: 308-317Crossref PubMed Scopus (74) Google Scholar). In fetal mice, a multilayered SC develops between days 17 and 19 (gestation is between days 19 and 20; see references in Hanley et al., 1997aHanley K. Devaskar V.P. Hicks S.J. et al.Hypothyroidism delays fetal stratum corneum development in mice.Ped Res. 1997; 42: 610-614Crossref PubMed Scopus (28) Google Scholar). Epidermal lipid synthesis peaks prior to barrier formation, declining as the barrier is formed (Hurt et al., 1995Hurt K. Hanley K. Williams M.L. Feingold K.R. Cutaneous lipid synthesis during late fetal development in the rat.Arch Dermat Res. 1995; 287: 754-760Crossref PubMed Scopus (22) Google Scholar), whereas the lipid processing enzymes, steroid sulfatase and β-glucocerebrosidase, increase in parallel with barrier formation (Hanley et al., 1997bHanley K. Jiang Y. Holleran W.M. Elias P.M. Williams M.L. Feingold K.R. Glucosylceramide metabolism is regulated during normal and hormonally-stimulated epidermal barrier development in the rat.J Lipid Res. 1997; 38: 576-584Abstract Full Text PDF PubMed Google Scholar, Hanley et al., 1997cHanley K. Jiang Y. Katagara C. Feingold K.R. Williams M.L. Steroid sulfatase and cholesterol sulfotransferase regulation during epidermal barrier ontogenesis in the fetal rat.J Invest Dermatol. 1997; 108: 871-875Abstract Full Text PDF PubMed Scopus (30) Google Scholar). Likewise, markers of epidermal terminal differentiation, such as involucrin and profilaggrin, peak later, coincident with barrier formation (Bickenbach et al., 1995Bickenbach J.R. Green J.M. Bundman B.S. Rothnagel J.A. Roop D.R. Loricrin expression is coordinated with other epidermal proteins and the appearance of lipid lamellar granules in development.J Invest Dermatol. 1995; 104: 405-410Abstract Full Text PDF PubMed Scopus (99) Google Scholar). Here, we asked first whether the epidermal Ca++ gradient forms in utero, despite being bathed in an isotonic milieu; second, whether changes in the Ca++ gradient correspond with, and account for, some or all of the developmental milestones described above. Our results show that: (i) a Ca++ gradient forms in utero, coincident with barrier ontogenesis; (ii) the Ca++ gradient is virtually absent in fetal rodent epidermis at a time when lipid synthesis is high, and lamellar body secretion appears accelerated; and (iii), conversely, a Ca++ gradient forms late in gestation, coincident with a decline in lipid synthesis, and in parallel with the generation of a multilayered SC and a functional barrier. Timed pregnant (plug date = day 0) Sprague-Dawley rats and SwissWebster mice were obtained from Simonsen Laboratories (Gilroy, CA). Maternal rats were anesthetized on gestational days 19, 20, and 21, and fetuses delivered prematurely by Cesarean section, whereas maternal mice were treated similarly on days 17, 18, 19, and 20. In addition, postnatal and adult epidermis were assessed, as indicated in the text and Figure 1(e)gends. We employed lanthanum perfusion, as described previously (Elias et al., 1981Elias P.M. Fritsch P. Lampe M. Williams M. Brown B. Nemanic M.K. Grayson S. Retinoid effects on epidermal structure, differentiation, and permeability.Lab Invest. 1981; 44: 531-540PubMed Google Scholar; Hanley et al., 1996Hanley K. Rassner U. Elias P.M. Williams M.L. Feingold K.R. Epidermal barrier ontogenesis: Maturation in serum-free media and acceleration by glucocorticoids and thyroid hormone but not selected growth factors.J Invest Dermatol. 1996; 106: 404-411Crossref PubMed Scopus (60) Google Scholar), to delineate the time course of barrier development in fetal mouse skin, because fetal mice are too small for routine transepidermal water loss measurements. Briefly, skin samples are incubated for 1 h in a 1:1 solution of 8% lanthanum nitrate (Electron Microscopy Sciences, Ft. Washington, PA) in 0.05 M Tris buffer and Karnovsky’s fixative (2% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer) at room temperature. Samples are then washed in cacodylate buffer, fixed further in half strength Karnovsky’s fixative overnight at 4°C, and transferred to ethanol solutions for dehydration and embedding. Ion capture cytochemistry was performed on fetal rat and mouse samples, as described in prior publications (e.g., Menon et al., 1985Menon G.K. Grayson S. Elias P.M. Ionic calcium reservoirs in mammalian epidermis: Ultrastructural localization by ion-capture cytochemistry.J Invest Dermatol. 1985; 84: 508-512Abstract Full Text PDF PubMed Scopus (390) Google Scholar). Briefly, the primary fixative contains 2% paraformaldehyde, 2.5% glutaraldehyde, 0.09 M potassium oxalate, 0.04 M sucrose, adjusted to pH 7.4. Samples are fixed overnight at 4°C, and then postfixed in 1% osmium tetroxide, containing 2% potassium pyroantimonate, pH 7.4 for 2 h at 4°C in the dark. Tissue samples are then washed in alkalinized distilled water (pH 10), and transferred to ethanol solutions for dehydration and embedding. Fetal rat and mouse skin samples were fixed in half strength Karnovsky’s fixative, postfixed in both osmium tetroxide and reduced ruthenium tetroxide (Hou et al., 1991Hou S.Y.E. Mitra A.K. White S.H. Menon G.K. Ghadially R. Elias P.M. Membrane structures in normal and essential fatty acid deficient stratum corneum: Characterization by ruthenium tetroxide staining and x-ray diffraction.J Invest Dermatol. 1991; 96: 215-223Abstract Full Text PDF PubMed Google Scholar), and embedded in an epoxy mixture. Ultra-thin sections, with and without additional contrasting (lead citrate/uranyl acetate), were examined in a Zeiss 10 A electron microscope operated at 60 kV. PIXE studies were performed using a modification of the methods of Bunse et al., 1991Bunse T. Steigleder G.K. Hofert M. Gonsier B. PIXE analysis in uninvolved skin of atopic patients and aged skin.Acta Dermato Vener. 1991; 71: 287-290PubMed Google Scholar. Four millimeter squared slice skin samples were collected from 18, 19, and 21 d old fetuses and from 1 or 2 d old pups and adult rats. Samples were frozen in liquid propane, transferred to liquid nitrogen, and stored at –50°C. After 30 μM sections were cut, they were transferred to nylon foils and freeze-dried for 12 h at –80°C. Samples were analyzed by microbeam particle induced X-ray emission, with beam currents of up to 900 pA, beam spatial resolution of 2–3 μm, and a beam energy of up to 3 MeV. X-rays were detected with a Si (Li) detector that subtended a solid angle of ≈100 msr. The detector was located at an angle of 135 with respect to the incident beam. Charge was collected in a biased Faraday cup located behind the sample. X-rays were recorded in list mode along with coincident beam spatial coordinates arising from scanning the beam electrostatically over the sample in a point by point raster mode. Data were reduced off-line so that X-ray spectra from subregions could be extracted from each irradiated region. X-ray spectra were analyzed with the PIXE spectrum fitting code (Antolak and Bench, 1994Antolak A.J. Bench G.S. The Liverrnore PIXE spectrum analysis package.Nucl Instr Meth. 1994; B90: 596-601Crossref Scopus (33) Google Scholar). A series of thin film calibration standards containing CA2+ were used to measure the efficiency of the X-ray detection system. To normalize the X-ray yields for variation in target thickness in order to obtain concentration data, scanning transmission ion microscopy was used to measure tissue projected densities. Each sample was measured in triplicate. Data are presented as the mean ± SD. Barrier function in rodents develops late in fetal developmentPrior studies in the fetal rat have shown that the appearance of mature lamellar unit structures in a multilayered SC corresponds with the appearance of a competent barrier by day 21, measured both by the ability of the SC to exclude lanthanum nitrate (Hanley et al., 1996Hanley K. Rassner U. Elias P.M. Williams M.L. Feingold K.R. Epidermal barrier ontogenesis: Maturation in serum-free media and acceleration by glucocorticoids and thyroid hormone but not selected growth factors.J Invest Dermatol. 1996; 106: 404-411Crossref PubMed Scopus (60) Google Scholar) and by a decrease in transepidermal water loss levels comparable with postnatal skin (Aszterbaum et al., 1992Aszterbaum M. Menon G.K. Feingold K.R. Williams M.L. Ontogeny of the epidermal barrier to water loss in the rat: correlation of function with stratum corneum structure and lipid content.Pediatr Res. 1992; 31: 308-317Crossref PubMed Scopus (74) Google Scholar). Because rat epidermis fails to generate a prominent Ca++ gradient (see below), we examined epidermal development in the fetal mouse, which displays a prominent Ca++ gradient. A distinct SC first appeared at day 17, and between days 17 and 18 the extracellular spaces at the SG–SC interface became engorged with secreted lamellar body contents Figure 1a. In contrast, the cytosol of the outermost granular cell was largely devoid of lamellar bodies, consistent with accelerated formation and secretion of these organelles. Mature lamellar membrane unit structures first appeared in a patchy distribution in the extracellular spaces of the SC by day 17–18 Figure 1b. Finally, abundant lamellar bodies again were present in the cytosol after day 18 (not shown; see Hanley et al., 1997aHanley K. Devaskar V.P. Hicks S.J. et al.Hypothyroidism delays fetal stratum corneum development in mice.Ped Res. 1997; 42: 610-614Crossref PubMed Scopus (28) Google Scholar). These studies show that fetal murine epidermis develops morphologic features of a competent barrier 1 or 2 d prior to birth, as does fetal rat epidermis. To determine whether these morphologic landmarks indicate the development of a competent barrier in the fetal mouse, we next examined the permeation of an electron-dense, water-soluble tracer, lanthanum nitrate, in the epidermis of fetal mice between days 15 and 19 of gestation. Whereas prior to day 17–18 (i.e., days 15–17) tracer freely permeated throughout the entire epidermis Figure 2a, after day 17–18 tracer was excluded from both the SG–SC interface and the extracellular spaces of the SC Figure 2b. These studies show that a competent barrier emerges in parallel with structural markers of SC development in fetal mouse skin, i.e., between days 17 and 18. We next asked whether the above-described, sequential changes in morphologic markers of barrier formation might be linked to changes in the epidermal Ca++ gradient Figure 3, 4. Whereas the neonatal mouse exhibits a prominent Ca++ gradient on ultrastructural cytochemistry Figure 3b, the neonatal rat displays much lower, absolute levels of Ca++ precipitates in the epidermis (not shown), making it difficult to evaluate changes in the Ca++ gradient in relation to fetal barrier development by ultrastructural cytochemistry. As seen in Figure 3a, the neonatal mouse displays no calcium gradient at days 16 and 17, a partial Ca++ gradient at day 18 (not shown), and a prominent Ca++ gradient at day 19 Figure 3bFigure 4PIXEshows development of the Ca++ gradient in rat epidermis late in fetal development. A distinct gradient is present as early as day 20, with further acceleration thereafter. Data are mean ± SD for three separate recordings from three different samples.View Large Image Figure ViewerDownload (PPT) The appearance of the latter is comparable with neonatal murine epidermis (Menon et al., 1985Menon G.K. Grayson S. Elias P.M. Ionic calcium reservoirs in mammalian epidermis: Ultrastructural localization by ion-capture cytochemistry.J Invest Dermatol. 1985; 84: 508-512Abstract Full Text PDF PubMed Scopus (390) Google Scholar); i.e., accumulation of extra- and intracellular Ca++ in the SG, and low visible levels in the SC and lower epidermal levels. Because the rat exhibits a less distinct Ca++ gradient than the mouse when examined by ultrastructural cytochemistry, we utilized an alternative, more sensitive and quantitative technique, PIXE, to measure changes in the epidermal Ca++ gradient during fetal rat development Figure 4. Moreover, our prior studies have shown good correlation between ion capture cytochemistry and PIXE when both were applied to the same samples (Mao-qiang et al., 1997Mao-qiang M. Mauro T. Bench G. Warren R. Elias P.M. Feingold K.R. Calcium and potassium inhibit barrier recovery after disruption, independent of the type of insult in hairless mice.Exp Dermatol. 1997; 6: 36-40Crossref PubMed Scopus (44) Google Scholar). Despite the low absolute levels of Ca++ in the epidermis (for comparison, peak Ca2+ levels in human epidermis are ∼400–500 mg per hg), an epidermal Ca++ gradient appears late in gestation (between days 20 and 21), coincident with barrier development in utero. Moreover, gradient formation is due to both a progressive decrease in Ca++ in the lower epidermis, and increased Ca++ levels in the outer epidermis. These results show, in two different species, and by two complementary techniques, that a Ca++ gradient appears in fetal epidermis in parallel with barrier development. These studies show that an epidermal Ca++ gradient develops late in gestation, parallel to the emergence of a competent permeability barrier. The epidermal Ca++ gradient, first recognized by ultrastructural cytochemistry (Menon et al., 1985Menon G.K. Grayson S. Elias P.M. Ionic calcium reservoirs in mammalian epidermis: Ultrastructural localization by ion-capture cytochemistry.J Invest Dermatol. 1985; 84: 508-512Abstract Full Text PDF PubMed Scopus (390) Google Scholar), has also been demonstrated by several quantitative methods in adult human and murine epidermis (Forslind, 1987Forslind B. Quantitative X-ray microanalysis of skin.Acta Derma Vener. 1987; 134: 1-8Google Scholar. In our most recent study, ion capture cytochemistry and PIXE provided very similar results (Mao-qiang et al., 1997Mao-qiang M. Mauro T. Bench G. Warren R. Elias P.M. Feingold K.R. Calcium and potassium inhibit barrier recovery after disruption, independent of the type of insult in hairless mice.Exp Dermatol. 1997; 6: 36-40Crossref PubMed Scopus (44) Google Scholar), further validating the application of either one or both methods to experimental systems. Because a prominent gradient is present in postnatal murine epidermis (Menon et al., 1985Menon G.K. Grayson S. Elias P.M. Ionic calcium reservoirs in mammalian epidermis: Ultrastructural localization by ion-capture cytochemistry.J Invest Dermatol. 1985; 84: 508-512Abstract Full Text PDF PubMed Scopus (390) Google Scholar), we utilized cytochemistry for the studies in fetal murine skin, and PIXE (a more sensitive and quantitative method) for studies in fetal rat skin, where the gradient is less prominent. Regardless of the method of barrier disruption, prior studies have shown the Ca++ gradient is lost (Menon et al., 1992aMenon G.K. Elias P.M. Feingold K.R. Localization of calcium in murine epidermis following disruption and repair of the permeability barrier.Cell Tiss Res. 1992; 270: 503-512Crossref PubMed Scopus (164) Google Scholar; Mao-qiang et al., 1997Mao-qiang M. Mauro T. Bench G. Warren R. Elias P.M. Feingold K.R. Calcium and potassium inhibit barrier recovery after disruption, independent of the type of insult in hairless mice.Exp Dermatol. 1997; 6: 36-40Crossref PubMed Scopus (44) Google Scholar h in parallel with barrier recovery. The reappearance of the Ca++ gradient during barrier recovery following acute insults, and the gradual appearance of a Ca++ gradient during fetal development, appear to be analogous in several respects. In postnatal epidermis, acute barrier disruption depletes the Ca++ gradient, provoking the en masse secretion of preformed lamellar bodies from the outermost SG cell (Menon et al., 1992aMenon G.K. Elias P.M. Feingold K.R. Localization of calcium in murine epidermis following disruption and repair of the permeability barrier.Cell Tiss Res. 1992; 270: 503-512Crossref PubMed Scopus (164) Google Scholar, Menon et al., 1992bMenon G.K. Feingold K.R. Elias P.M. The lamellar body secretory response to barrier disruption.J Invest Dermatol. 1992; 98: 279-289Abstract Full Text PDF PubMed Scopus (249) Google Scholar. Shortly thereafter, cholesterol and fatty acid synthesis increase; and nascent lamellar bodies are generated and secreted in an accelerated fashion (reviewed in Feingold, 1991Feingold K.R. The regulation and role of epidermal lipid synthesis.Adv Lipid Res. 1991; 24: 57-79Crossref PubMed Google Scholar; Elias, 1996Elias P.M. Stratum corneum architecture, metabolic activity, and interactivity with subjacent cell layers.Exp Dermatol. 1996; 5: 191-201Crossref PubMed Scopus (110) Google Scholar). In parallel with the reappearance of the Ca++ gradient, both lipid synthesis and lamellar body secretion slow down. A similar, if not identical relationship appears to pertain during fetal barrier development. We also showed previously that lipid synthesis peaks prior to barrier formation (Hurt et al., 1995Hurt K. Hanley K. Williams M.L. Feingold K.R. Cutaneous lipid synthesis during late fetal development in the rat.Arch Dermat Res. 1995; 287: 754-760Crossref PubMed Scopus (22) Google Scholar), at a time when no demonstrable gradient is present (these studies), and synthesis rates decline as the barrier is formed. Moreover, we showed here a potential relationship between the status of the Ca++ gradient and the cytosolic pool of lamellar bodies, i.e., secretion appears to accelerate prior to barrier formation, whereas large numbers of lamellar bodies reappear coincident with gradient and barrier formation, implying slowed secretion rates. The declining rates of lipid accumulation in the SC late in barrier development (Aszterbaum et al., 1992Aszterbaum M. Menon G.K. Feingold K.R. Williams M.L. Ontogeny of the epidermal barrier to water loss in the rat: correlation of function with stratum corneum structure and lipid content.Pediatr Res. 1992; 31: 308-317Crossref PubMed Scopus (74) Google Scholar), in the face of maintenance secretion rates, can be explained by the continued expansion and retention of the SC compartment in utero. Because artificial restoration of the barrier with a vapor-impermeable wrap blocks reformation of the Ca++ gradient (Menon et al., 1992aMenon G.K. Elias P.M. Feingold K.R. Localization of calcium in murine epidermis following disruption and repair of the permeability barrier.Cell Tiss Res. 1992; 270: 503-512Crossref PubMed Scopus (164) Google Scholar), it seems likely that the gradient forms passively as ions are trapped under a competent barrier, which would minimize net water movement and prevent escape of ions. Yet, in both of the fetal rodent models, a Ca++ gradient forms despite exposure of the outer epidermis to an isotonic milieu, where water movement should be minimal, suggesting a role for active mechanism(s) in Ca++ gradient formation. Moreover, an active rather than a passive mechanism is suggested by the PIXE data shown here, which show a reduction in Ca++ levels in the lower epidermis, rather than a progressive accumulation of Ca++ in the outer epidermis during development. Alternatively, the Ca++ gradient could form passively as Ca++ is sequestered by calcium binding proteins, recently demonstrated to be present in amniotic fluid (Hitomi et al., 1996Hitomi J. Yamaguchi K. Kikuchi Y. Kimura T. Marnyama K. Nagasaki K. A novel calcium-binding protein in amniotic fluid, CAAF1: its molecular cloning and tissue distribution.J Cell Sci. 1996; 109: 805-815Crossref PubMed Google Scholar). At this point, direct evidence for either active or passive mechanisms in epidermal ion gradient formation is lacking. Finally, fetal barrier development can be divided conceptually into two overlapping stages Figure 5. During the initial phases of barrier development, epidermal cholesterol, fatty acid, and ceramide synthesis peak, and lamellar bodies form and are rapidly secreted (shown previously for the fetal rat and here for the fetal mouse). At this stage, epidermal differentiation is still rudimentary, mature lamellae are not present in the SC interstices, enzymes related to lamellar processing are expressed at low levels, and a competent barrier is not yet present. In contrast, during the later stage of barrier development, lipid synthesis slows down, and lamellar body formation and secretion decrease to normal (i.e., maintenance) levels. Simultaneously, epidermal differentiation proceeds (Bickenbach et al., 1995Bickenbach J.R. Green J.M. Bundman B.S. Rothnagel J.A. Roop D.R. Loricrin expression is coordinated with other epidermal proteins and the appearance of lipid lamellar granules in development.J Invest Dermatol. 1995; 104: 405-410Abstract Full Text PDF PubMed Scopus (99) Google Scholar), mature extracellular lamellae form, expression of processing enzymes increases, and a competent barrier emerges. Prior studies have shown that lamellar body secretion accelerates in low Ca++ , whereas physiologic Ca++ inhibits cholesterol synthesis and lamellar body secretion. Moreover, a large number of in vitro studies have shown that several key steps in the terminal epidermal differentiation program require a high extracellular Ca++ level (Hennings et al., 1983Hennings H. Holbrook K.A. Yuspa S.H. Factors influencing calcium-induced terminal differentiation in cultured mouse epidermal cells.J Cellular Physiol. 1983; 116: 265-281Crossref PubMed Scopus (92) Google Scholar; Yuspa et al., 1989Yuspa S.H. Kilkenny A.K. Steinert P.M. Roop D.R. Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro.J Cell Biol. 1989; 109: 1207-1217Crossref PubMed Scopus (514) Google Scholar). Because the above-described processes are regulated by changes in ambient Ca++ concentrations, it is tempting to link each of them to the observed changes in the Ca++ gradient Figure 5; however, other experimental approaches will be required to determine which of these developmental milestones are regulated directly by changes in the Ca++ gradient. This work was supported by NIH grants AR19098, AR39369, AR39448 (PP), HD29706, and the Medical Research Service, Veteran Affairs Medical Center. This work was partially performed under the auspices of the US Department of Energy by the Lawrence Livermore National Laboratory under contract W-7405-EnG-48. Sue Allen and Lee Wong capably prepared the manuscript.
Referência(s)