Biomedical subjects

T Mauro

Publications and source records attributed to T Mauro.

At least 19 recordsLinked to original sources

PubMed2006

Hair cycle and wound healing in mice with a keratinocyte-restricted deletion of FAK.

Focal adhesion kinase (FAK) is a critical component in transducing signals downstream of both integrins and growth factor receptors. To determine how the loss of FAK affects the epidermis in vivo, we have generated a mouse model with a keratinocyte-restricted deletion of fak (FAKK5 KO mice). FAK(K5 KO) mice displayed three major phenotypes--irregularities of hair cycle, sebaceous glands hypoplasia, and a thinner epidermis--pointing to defects in the proliferative capacity of multipotent stem cells found in the bulge. FAK-null keratinocytes in conventional primary culture undergo massive apoptosis hindering further analyses, whereas the defects observed in vivo do not shorten the mouse lifespan. These results suggest that the structure and the signaling environment of the native tissue may overcome the lack of signaling through FAK. Our findings point to the importance of in vivo and three-dimensional in vitro models in analyses of cell migration, proliferation, and survival. Surprisingly, the difference between FAKloxP/+ and FAKK5 KO mice in wound closure was not statistically significant, suggesting that in vivo loss of FAK does not affect migration/proliferation of basal keratinocytes in the same way as it affects multipotent stem cells of the skin.

Animals↗

PubMed2002

The ENaC channel is required for normal epidermal differentiation.

Ionic fluxes are important for critical aspects of keratinocyte differentiation, including synthesis of differentiation-specific proteins, enzymatic catalysis of protein cross-linking, post-transcriptional processing of profilaggrin, and lipid secretion. The epithelial sodium channel is expressed in epidermis and the expression of its alpha and beta subunits is enhanced as keratinocytes differentiate. In order to ascertain the role of the epithelial sodium channel in epidermal differentiation, we examined skin of mice in which the epithelial sodium channel alpha subunit had been deleted. Newborn -/- mice, in which the alpha subunit had been completely inactivated, demonstrated epithelial hyperplasia, abnormal nuclei, premature secretion of lipids, and abnormal keratohyaline granules. In addition, immunohistochemistry demonstrated that expression of the differentiation markers K1, K6, and involucrin were abnormal. These data suggest that the epithelial sodium channel modulates ionic signaling for specific aspects of epidermal differentiation, such as synthesis or processing of differentiation- specific proteins, and lipid secretion.

Animals↗

PubMed2000

The calcium sensing receptor and its alternatively spliced form in murine epidermal differentiation.

We have recently reported that human keratinocytes express both the full-length calcium sensing receptor (CaR) and an alternatively spliced form lacking exon 5, which were suggested to be involved in calcium induced keratinocyte differentiation. To understand further the role of these CaRs, we analyzed the structure of mouse CaRs, and investigated their role using a mouse model in which only the full-length CaR was disrupted. Our results show that both the full-length and the alternatively spliced variant lacking exon 5 encoding 77 amino acids of the extracellular domain were expressed in mouse epidermis. The deletion of the full-length CaR increased the production of the alternatively spliced form of CaR in mutant mice. The keratinocytes derived from these mutant mice did not respond to extracellular calcium, suggesting that the full-length CaR is required to mediate calcium signaling in the keratinocytes. The loss of the full-length CaR altered the morphologic appearance of the epidermis and resulted in a reduction of the mRNA and protein levels of the keratinocyte differentiation marker, loricrin. These results indicate that CaR is important in epidermal differentiation, and that the alternatively spliced form does not fully compensate for loss of the full-length CaR.

Alternative Splicing↗

PubMed2000

Changes in HOXB6 homeodomain protein structure and localization during human epidermal development and differentiation.

HOX homeodomain proteins are master developmental regulators, which are now thought to function as transcription factors by forming cooperative DNA binding complexes with PBX or other protein partners. Although PBX proteins exhibit regulated subcellular localization and function in the nucleus in other tissues, little data exists on HOX and PBX protein localization during skin development. We now show that the HOXB6 protein is expressed in the suprabasal layer of the early developing epidermis and throughout the upper layers of late fetal and adult human skin. HOXB6 signal is cytoplasmic throughout fetal epidermal development, but substantially nuclear in normal adult skin. HOXB6 protein is also partially nuclear in hyperproliferative skin conditions, but appears to be cytoplasmic in basal and squamous cell carcinomas. Although all three PBX genes are expressed in fetal epidermis, none of the three PBX proteins exhibit nuclear co-localization with HOXB6 in either fetal or adult epidermis. RNA and protein data suggest that a truncated HOXB6 protein, lacking the homeodomain, is expressed in undifferentiated keratinocytes and that the full-length protein is induced by differentiation. GFP-fusion proteins were used to demonstrate that the full-length HOXB6 protein is localized to the nucleus while the truncated protein is largely cytoplasmic. Taken together, these data suggest that during epidermal development the truncated HOXB6 isoform may function by a mechanism other than as DNA binding protein, and that most of the nuclear, homeodomain-containing HOXB6 protein does not utilize PBX proteins as DNA binding partners in the skin. Published 2000 Wiley-Liss, Inc.

Adult↗

PubMed2000

Mutations in ATP2C1, encoding a calcium pump, cause Hailey-Hailey disease.

Hailey-Hailey disease (HHD, MIM 16960) is inherited in an autosomal dominant manner and characterized by persistent blisters and erosions of the skin. Impaired intercellular adhesion and epidermal blistering also occur in individuals with pemphigus (which is due to autoantibodies directed against desmosomal proteins) and in patients with Darier disease (DD, MIM 124200), which is caused by mutations in a gene encoding a sarco/endoplasmic reticulum (ER)-Golgi calcium pump. We report here the identification of mutations in ATP2C1, encoding the human homologue of an ATP-powered pump that sequesters calcium into the Golgi in yeast, in 21 HHD kindreds. Regulation of cytoplasmic calcium is impaired in cultured keratinocytes from HHD patients, and the normal epidermal calcium gradient is attenuated in vivo in HHD patients. Our findings not only provide an understanding of the molecular basis of HHD, but also underscore the importance of calcium control to the functioning of stratified squamous epithelia.

Adult↗

PubMed1999

Epithelial sodium channels are upregulated during epidermal differentiation.

Terminal differentiation of epidermal keratinocytes is linked to transmembrane ion flux. Previously, we have shown that amiloride, an inhibitor of epithelial sodium channels, blocks synthesis of differentiation-specific proteins in normal human keratinocytes. Here, we have identified the specific subunits of amiloride-sensitive human epithelial sodium channels in relation to differentiation of cultured human keratinocytes, as well as to epidermal development. As assessed by northern hybridization, RNase protection assay, and reverse transcription-polymerase chain reaction, transcripts encoding functional alpha and regulatory beta subunits of human epithelial sodium channels were expressed both in cultured keratinocytes and in epidermis at levels comparable with the kidney. The mRNA expression of both human epithelial sodium channel-alpha and -beta increased during calcium-induced keratinocyte differentiation. Whereas the beta subunit of human epithelial sodium channel was induced by elevated concentrations of calcium, the alpha subunit increased with duration of culture. The regulatory gamma subunit was less abundant but also expressed in epidermis. Both human epithelial sodium channel-alpha and -beta were localized throughout the nucleated layers of human adult epidermis, but these channels were not detected in early stages of fetal epidermal development. This co-ordinated expression of subunits suggests that epithelial sodium channels may play an important part in both epidermal differentiation and skin development, presumably by modulating ion transport required for epidermal terminal differentiation.

Blotting, Western↗

PubMed1999

Using lasers in diabetic wound healing.

Lasers are a treatment choice that appeals to patients. Early research suggests that laser therapy may have a role in hastening wound healing. Attempts have been made to use helium neon, CO2, and KTP lasers in encouraging wound healing in diabetics. We will review the English literature related to laser use in diabetic wound healing and discuss the concept of the use of nondestructive lasers for biostimulation. Further research is needed to assess effectiveness of biostimulation for diabetic wound healing.

Animals↗

PubMed1998

Barrier recovery is impeded at neutral pH, independent of ionic effects: implications for extracellular lipid processing.

Epidermal permeability barrier homeostasis requires the postsecretory processing of polar lipid precursors into nonpolar lipid products within the stratum corneum (SC) interstices by a family of lipid hydrolases. A specific requirement for beta-glucocerebrosidase (beta-GlcCer'ase), which exhibits a distinct acidic pH optimum, is particularly well documented. Therefore, we sought to determine whether the recovery of the barrier after acute insults requires acidification of the SC. We examined permeability barrier recovery by assessing changes in transepidermal water loss (TEWL), SC membrane ultrastructure utilizing ruthenium tetroxide (RuO4) postfixation, and beta-GlcCer'ase activity by in situ zymography at an acidic vs neutral pH. Barrier recovery proceeded normally when acetone-treated skin was exposed to solutions buffered to an acidic pH. In contrast, the initiation of barrier recovery was slowed when treated skin was exposed to neutral or alkaline pH, regardless of buffer composition. In addition, enhancement of the alkaline buffer-induced delay in barrier recovery occurred with Ca2+ and K+ inclusion in the buffer. Moreover, the pH-dependent alteration in barrier recovery appeared to occur through a mechanism that was independent of Ca(2+)- or K(+)-controlled lamellar body secretion, since both the formation and secretion of lamellar bodies proceeded comparably at pH 5.5 and pH 7.4. In contrast, exposure to pH 7.4 (but not pH 5.5) resulted in both the persistence of immature, extracellular lamellar membrane structures, and a marked decrease in the in situ activity of beta-GlcCer'ase. These results suggest first that an acidic extracellular pH is necessary for the initiation of barrier recovery, and second that the delay in barrier recovery is a consequence of inhibition of postsecretory lipid processing.

Animals↗

PubMed1998

The secretory granular cell: the outermost granular cell as a specialized secretory cell.

The contents of epidermal lamellar bodies (LB) are delivered selectively to the intercellular spaces at the stratum granulosum (SG)-stratum corneum (SC) interface. We assessed the subcellular basis for LB secretion first by confocal microscopy, following labeling with Nile red or NBD-ceramide, which reveals a tubulo-reticular membrane system within the apical cytosol of the outermost SG cell layer under basal conditions, changing to a more peripheral staining pattern when secretion is stimulated. Ultrastructural study demonstrates that this network is composed of a widely disbursed trans-Golgi-like network (TGN), associated with arrays of contiguous LB, and deep invaginations of the SG-SC interface. Under basal conditions, limited fusion of apically directed LB leads to deep, interconnected invaginations of the apical plasma membrane, resulting in the formation of an extensive, honeycomb extension of the SG-SC interface. Still deeper invaginations and more extensive organelle fusion develop after the epidermis is acutely permeabilized by either acetone treatment, sonophoresis, or iontophoresis. Finally, nascent LB appear to bud off cisternae of the TGN, a process that appears to accelerate after barrier disruption. The deep invaginations of the SG-SC interface; the wide distribution of the TGN within the apical cytosol; the association of nascent LB with the TGN; and the rapid fusion of LB with these invaginations, deep within the cytosol, account for (i) the polarized secretion of LB from the apex of the outermost SG cell, and (ii) the rapid LB-secretory response to barrier perturbations. Finally, our results point to the outermost SG cell as a uniquely specialized secretory cell. We propose the term "secretory granulocyte" to encompass the specialized features of these cells.

4-Chloro-7-nitrobenzofurazan↗

PubMed1998

Formation of the epidermal calcium gradient coincides with key milestones of barrier ontogenesis in the rodent.

The epidermal permeability barrier forms late in gestation, coincident with decreased lipid synthesis, increased lipid processing, and development of a mature, multi-layered 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.

Animals↗

PubMed1998

Acute barrier perturbation abolishes the Ca2+ and K+ gradients in murine epidermis: quantitative measurement using PIXE.

Epidermal permeability barrier homeostasis requires the delivery of lipids and hydrolytic enzymes by lamellar body exocytosis from the uppermost granular cells, a process that is upregulated following barrier disruption. As lamellar body secretion is controlled by ionic concentrations, especially Ca2+ and K+, we used a quantitative technique, microbeam proton-induced X-ray emission, to measure Ca2+, K+, Cl-, and P concentrations before and after acute barrier perturbation by acetone applications. We found a steep gradient of Ca2+ in normal tissue, peaking in the outer stratum granulosum, which disappeared after barrier disruption, and partially reformed as the barrier recovered. A similar gradient, peaking somewhat lower in the epidermis (i.e., at the stratum granulosum-stratum corneum interface), was found for K+. Epidermal concentrations of K+ also decreased after barrier abrogation, although to a lesser extent than Ca2+. In contrast, P and Cl- demonstrated distribution gradients at baseline, which remained unchanged after barrier disruption. These studies quantitate the levels of Ca2+, K+, Cl-, and P within specific epidermal cell layers at baseline, and in relation to changes in permeability barrier integrity. Ca2+ and K+, but not Cl- or P, decrease after barrier disruption, consistent with these two ion's role in barrier repair.

Animals↗

PubMed1997

Alternatively spliced forms of the cGMP-gated channel in human keratinocytes.

Alternatively spliced forms of the alpha subunit of the cGMP-gated channel have been cloned from human keratinocytes. One form encodes a complete channel which is almost identical to the rod photoreceptor. A second spliced variant would encode a protein missing a portion of the intracellular hydrophilic domain and the putative first transmembrane domain. Both complete and spliced variants of the channel also were found in epidermis. The expression of the complete form of the channel was induced by levels of extracellular calcium which promote keratinocyte differentiation. The cGMP-gated channel may play an important role in calcium induced keratinocyte differentiation by mediating Ca2+ entry.

Alternative Splicing↗

PubMed1997

Keratinocyte K+ channels mediate Ca2+-induced differentiation.

K+ channel activation has been associated with growth or differentiation in many cells. We have previously identified a 70-pS K+ channel that was found only in differentiated involucrin-positive cells. In this study we examined the role of K+ channels in Ca2+-induced keratinocyte differentiation. Consistent with our previous report, we found that a K+ conductance developed only in cells cultured in high extracellular Ca2+. Addition of charybdotoxin or verapamil blocked these K+ channels and inhibited Ca2+-induced differentiation, as assessed by cornified envelope formation or transglutaminase activity. These results suggest that K+ channel activation is necessary for Ca2+-induced differentiation. Finally, we used (125)I-labeled charybdotoxin to demonstrate the presence of K+ channels in intact human and mouse epidermis, hair follicles, and eccrine glands, indicating that these channels are found in keratinocytes both in vitro and in vivo. Thus K+ channels may moderate Ca2+ influx in more differentiated keratinocytes and may play a central role in keratinocyte differentiation.

Animals↗

PubMed1997

Calcium and potassium inhibit barrier recovery after disruption, independent of the type of insult in hairless mice.

Disruption of the cutaneous permeability barrier induces metabolic responses in the epidermis which result in barrier recovery. Barrier disruption by either solvent treatment or tape stripping results in the loss of the epidermal calcium gradient. Previous studies in acetone treated hairless mice have shown that maintaining this calcium gradient inhibits barrier repair, suggesting that alterations in the epidermal calcium concentration may be an important signal for barrier homeostasis. In the present study, we show that in hairless mice disruption of the barrier by treatment with the detergent, SDS, also results in the loss of the calcium gradient, as demonstrated both semi-quantitatively with ultrastructural cytochemical localization and quantitatively using proton induced X-ray emission (PIXE). Additionally, immersion in calcium containing solutions delays barrier repair after either detergent (SDS treatment) or mechanical (tape stripping) disruption of the barrier, as reported previously for acetone treated skin. These results indicate that barrier disruption, regardless of the insult, induces changes in the epidermal calcium gradient which may play an important role in signaling the metabolic changes required for barrier homeostasis.

Animals↗

PubMed1996

Changes in calcium responsiveness and handling during keratinocyte differentiation. Potential role of the calcium receptor.

Extracellular calcium concentrations (Cao) > 0.1 mM are required for the differentiation of normal human keratinocytes in culture. Increments in Cao result in acute and sustained increases in the intracellular calcium level (Cai), postulated to involve both a release of calcium from intracellular stores and a subsequent increase in calcium influx through nonspecific cation channels. The sustained rise in Cai appears to be necessary for keratinocyte differentiation. To understand the mechanism by which keratinocytes respond to Cao, we measured the acute effects of Cao on Cai and calcium influx in keratinocytes at various stages of differentiation. We then demonstrated the existence of the calcium receptor (CaR) in keratinocytes and determined the effect of calcium-induced differentiation on its mRNA levels. Finally, we examined the role of Cai in regulating both the initial rise in Cai after the switch to higher Cao and the activity of the nonspecific cation channel through which calcium influx occurs. Our data indicate that the acute Cai response to Cao is lost as the cells differentiate and increase their basal Cai. These data correlated with the decrease in CaR mRNA levels in cells grown in low calcium. However, calcium influx as measured by 45Ca uptake increased with differentiation in 1.2mM calcium, consistent with the increase in CaR mRNA in these cells as well as the calcium-induced opening of the nonspecific cation channels. We conclude that the keratinocyte contains a CaR that regulates both the initial release of Cai from intracellular stores and the subsequent increase in calcium flux through nonspecific calcium channels. A rising level of Cai may turn off the release of calcium from intracellular stores while potentiating the influx through the nonspecific cation channels. Differentiation of keratinocytes appears to increase the CaR, which may facilitate the maintenance of the high Cai required for differentiation.

Animals↗

PubMed1995

Amiloride blocks a keratinocyte nonspecific cation channel and inhibits Ca(++)-induced keratinocyte differentiation.

Proliferation and differentiation in many cells are linked to specific changes in transmembrane ion fluxes. Previously, we have identified a nonspecific cation channel in keratinocytes, which is permeable to and activated by Ca++. To test whether this cation channel might serve as a pathway for Ca++ entry, we examined the effect of blocking this channel on membrane currents, markers of differentiation, and intracellular Ca++. In patch clamp studies, 10(-8) to 10(-6) M amiloride decreased the single-channel open probability. The same concentrations of amiloride inhibited the calcium-induced formation of cornified envelopes and activity of transglutaminase in a dose-dependent fashion. Amiloride inhibited the long-term rise of intracellular Ca++ induced by raised extracellular Ca++, without blocking the initial increase of intracellular Ca++. Amiloride at concentrations of 10(-7) to 10(-3) M did not change the resting intracellular pH of keratinocytes, although concentrations of 10(-6) M or greater inhibited the recovery from NH4(+)-induced acidification. To test whether the effect of amiloride was toxic, we measured DNA synthesis in the presence or absence of amiloride. DNA synthesis was unchanged, suggesting that amiloride's actions were not due to toxic effects. Although the exact mechanisms of amiloride's action remains to be determined, these experiments suggest that this compound may inhibit keratinocyte differentiation by blocking the nonspecific cation channel.

Amiloride↗

PubMed1995

Treatment of leg ulcers with cultured epithelial autografts: clinical study and case reports.

Leg ulcers are a significant medical problem in the United States. The number of affected patients at any given time is staggering. Treatment modalities have widely varied and are often unsuccessful. We evaluated the effectiveness of cultured epithelial autografts in the treatment of leg ulcers due to various etiologies that had failed other standard therapy. Eighty-six ulcers in 36 patients were treated with cultured epithelial autografts after full evaluation of their ulcers. The ulcers were grouped by etiology and healing was assessed by time to healing, number of ulcers healed and mean number of graft applications. The results achieved in the past five years have shown that cultured epithelial autografts are highly effective in treating chronic leg ulcers. Eighty percent of venous insufficiency ulcers healed in 5.7 weeks. Similar results were seen with ulcers of other etiologies including arterial insufficiency, pyoderma gangrenosum, scleroderma, and vasculitis. Positive results were seen in cases where other treatment methods had failed and seems independent of systemic corticosteroids.

Adult↗

PubMed1994

A role for ions in barrier recovery after acute perturbation.

The epidermal cutaneous permeability barrier can be disrupted by treatment with topical solvents. Recent studies have shown that barrier recovery, measured by the recovery of transepidermal water loss towards normal, is inhibited by high extracellular Ca++ and K+, and accelerated by low extracellular concentrations of these ions. To examine the effects of Ca++ or K+ fluxes on barrier recovery, we tested the effects on transepidermal water loss recovery of agents that modify these fluxes. K+ channel agonists or blockers modified the inhibitory effects on barrier recovery induced by raised extracellular Ca++ and K+. In addition, Na+/K+ adenosine 5' triphosphatase inhibitors reversed the inhibitory effects of high extracellular Ca++ and K+. Our results suggest that barrier recovery requires both Ca++ and K+ fluxes and are consistent with the hypothesis that both verapamil or dihydropyridine-sensitive Ca++-permeable channels and Ca++-sensitive K+ channels participate in epidermal permeability barrier homeostasis.

Animals↗