Cultured Human Keratinocytes Research Articles

Rapid culture of human keratinocytes in an autologous, feeder-free system with a novel growth medium

Background aimsThe ability to culture human keratinocytes is beneficial in the treatment of skin injury and disease, as well as for testing chemicals in vitro as a substitute for animal testing. ResultsWe have identified a novel culture medium for the rapid growth of keratinocytes from human skin. “Kelch's medium” supports keratinocyte growth that is as rapid as in the classical Rheinwald and Green method, but without the need for cholera toxin or xenogeneic feeder cells. It enables keratinocytes to out-compete co-cultured autologous fibroblasts so that separation of the epidermis from the dermis is no longer required before keratinocyte culture. Enzymatic digests of whole human skin can therefore be used to generate parallel cultures of autologous keratinocytes, fibroblasts and melanocytes simply by using different cell culture media. ConclusionsThis new keratinocyte medium and the simplified manufacturing procedures it enables are likely to be beneficial in skin engineering, especially for clinical applications.

Development of Keratinocyte Cell Lines Containing Extrachromosomal Human Papillomavirus Genomes.

Human papillomaviruses (HPVs) cause persistent infections in stratified cutaneous and mucosal epithelia. In these infections, the viral DNA replicates as low-copy-number, extrachromosomal, double-stranded-DNA circular plasmids in the nucleus of the dividing basal cells. When the infected cells begin the process of differentiation, the viral DNA amplifies to a high copy number and virions are assembled in the superficial cells. To study HPV DNA replication, our laboratory generates primary keratinocyte cell lines that contain replicating extrachromosomal HPV genomes. Here, we describe protocols to culture human keratinocytes, to transfect viral DNA into cells using electroporation, to determine the efficiency of genome establishment in cells with a colony-forming assay, and to measure the copy number and extrachromosomal status of viral genomes using Southern blotting. These methods can be used to study DNA replication of different oncogenic Alphapapillomavirus HPV types. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Electroporation to transfect keratinocytes with recircularized HPV genomes Alternate Protocol: Use of HPV replicon containing selection marker in keratinocyte transfection Support Protocol 1: Rheinwald-Green method of co-culture of irradiated J2 3T3 feeders and human keratinocytes Support Protocol 2: Recircularization of HPV genomes Basic Protocol 2: Quantitative colony formation assay to measure the efficiency of HPV genome establishment Basic Protocol 3: Southern blot analysis of extrachromosomal viral DNA Support Protocol 3: Hirt extraction of low-molecular-weight DNA Support Protocol 4: Qiagen DNeasy Blood & Tissue DNA extraction Support Protocol 5: Generation of a 32 P-labeled HPV DNA probe.

245 - Development of a keratinocyte-specific defined media suitable for self-assembled bilayered skin substitute production

Skin epithelial cell (keratinocytes) culture and self-assembled skin substitutes (SASS) have greatly improved treatments for patients with severe burns. To properly culture human keratinocytes, fetal bovine serum (FBS) must be added to the medium. However, FBS exact biological composition is undefined and is known for batch-to-batch variations. FBS is also associated with a risk of xenogeneic pathogen transmission. For these reproducibility and safety reasons, discarding FBS usage in both research and clinical contexts is desirable. Although keratinocyte-specific serum-free (defined) media are commercially available, they are not effective in culturing keratinocytes for more than a few passages and are not suitable for SASS production. Therefore, our objective is to develop a defined medium optimized for keratinocyte expansion and SASS production. Three candidate molecular factors capable of substantially promoting keratinocyte growth were initially identified from an experimental screening on a single cell population. This led to the development of an effective defined medium for culturing keratinocytes (DMK). DMK was then optimized in terms of cost-effectiveness by testing different concentrations of the factors previously identified, and three DMK derivatives (α, β, and γ) were formulated. Using two additional populations, DMK and its derivatives were ranked over their ability to sustain culture quality. Daily population doublings, passage count, and cell size were used as proxies. Keratinocytes cultured in DMKβ were smaller and could be grown over at least as many passages as in FBS-containing medium and more passages than those cultured in DMK or its other derivatives. Also, keratinocyte population doubling rates were greater in DMKβ. Keratinocyte cultured in either DMKβ or FBS-containing medium were morphologically comparable. SASS were then produced with both DMKβ and FBS-containing medium using four anatomically distinct keratinocyte populations. A portion of the amplified keratinocytes were also used to compare complete transcriptomic profiles. Substitute histological analyses showed no clear differences between those produced with DMKβ or FBS-containing medium. Substitute similarity was further evaluated with immunolabelling of K10, K14, K19, Ki67, filaggrin, and type IV collagen. In conclusion, this new defined medium is promising for keratinocyte culture and skin substitute production for research and clinical purposes.

Water Extract of Gromwell (Lithospermum erythrorhizon) Enhances Migration of Human Keratinocytes and Dermal Fibroblasts with Increased Lipid Synthesis in an in vitro Wound Scratch Model

Background: Although organic extracts of gromwell (Lithospermum erythrorhizon) have been shown to promote wound healing, the wound healing effects of water extracts of gromwell (WG) that are commonly used in traditional remedies have not been elucidated. Objective: We investigated whether WG promotes the migration and/or proliferation of cultured human keratinocytes (CHK) or dermal fibroblasts in parallel with increases in lipid synthesis during in vitro wound healing. Methods: CHK or fibroblasts were treated with 1–1,000 µg/ml WG for up to 48 h following scratch wound formation. Cell migration was assessed by measuring coverage (in percent) from the wound margin, while cell proliferation and lipid synthesis were determined by [³H]thymidine incorporation into DNA fractions, and [³H]palmitate or [³H]serine incorporation into lipid fractions, respectively. Results: Low-dose WG (1 µg/ml) enhanced the wound coverage for both CHK and fibroblasts at 24 h, while cell proliferation was not altered in either cell types. Synthesis of both total lipids and individual lipid classes, including phospholipids, sphingolipids and neutral lipids, were found to be increased at 24 h in CHK treated with 1 µg/ml WG; in similarly treated fibroblasts, only the syntheses of sphingolipids (such as ceramides and glucosylceramides), but not other lipid species, were significantly increased. In contrast, a higher dose of WG (10–1,000 µg/ml) did not enhance wound coverage, and 100 µg/ml WG neither altered cell proliferation nor lipid synthesis in both CHK and fibroblasts. Conclusion: Low-dose WG (1 µg/ml) enhances the migration of both CHK and fibroblasts with increased lipid synthesis in an in vitro wound scratch model.

PPARgamma activators stimulate aquaporin 3 expression in keratinocytes/epidermis

Aquaporin 3 (AQP3), a member of the aquaglyceroporin family, which transports water and glycerol, is robustly expressed in epidermis and plays an important role in stratum corneum hydration, permeability barrier function and wound healing. PPAR and LXR activation regulates the expression of many proteins in the epidermis and thereby can affect epidermal function. Here, we report that PPARgamma activators markedly stimulate AQP3 mRNA expression in both undifferentiated and differentiated cultured human keratinocytes (CHKs). The increase in AQP3 mRNA by PPARgamma activator occurs in a dose- and time-dependent fashion. Increased AQP3 mRNA levels are accompanied by an increase in AQP3 protein in undifferentiated keratinocytes and a significant increase in glycerol uptake. Activation of LXR, RAR and RXR also increases AQP3 mRNA levels in undifferentiated and differentiated CHKs, but to a lesser extent. PPARdelta activation stimulates AQP3 expression in undifferentiated CHKs but decreases expression in differentiated CHKs. In contrast, PPARalpha activators do not alter AQP3 expression. AQP9 and AQP10, other members of aquaglyceroporin family, are less abundantly expressed in CHKs, and their expression levels are not significantly altered by treatment with LXR, PPAR, RAR or RXR activators. Finally, when topically applied, the PPARgamma activator, ciglitazone, induces AQP3 but not AQP9 gene expression in mouse epidermis. Our data demonstrate that PPAR and LXR activators stimulate AQP3 expression, providing an additional mechanism by which PPAR and LXR activators regulate epidermal function.

Expression and regulation of GPAT isoforms in cultured human keratinocytes and rodent epidermis

Phospholipids are required for epidermal lamellar body formation. Glycerol 3-phosphate acyltransferases (GPATs) catalyze the initial step in the biosynthesis of glycerolipids. Little is known about the expression and regulation of GPATs in epidermis/keratinocytes. Here, we demonstrate that GPAT 1, 3, and 4 are expressed in epidermis/keratinocytes, whereas GPAT2 is not detected. In mouse epidermis, GPAT 3 and 4 are mainly localized to the upper layers whereas GPAT1 is found in both the upper and lower layers. GPAT1 and 3 mRNA increase during fetal rat epidermal development. No change in GPAT expression was observed in adult mice following acute permeability barrier disruption. Calcium-induced human keratinocyte differentiation increased GPAT3 mRNA whereas both GPAT1 and 4 mRNA levels decreased. In parallel, total GPAT activity increased 2-fold in differentiated keratinocytes attributable to an increase in N-ethylmaleimide (NEM) sensitive GPAT activity localized to microsomes with little change in NEM resistant activity, consistent with an increase in GPAT3. Furthermore, PPARγ or PPARδ activators increased GPAT3 mRNA, microsomal GPAT activity, and glycerol lipid synthesis without affecting the expression of GPAT1 or 4. Finally, both PPARγ and PPARδ activators increased GPAT3 mRNA via increasing its transcription. Thus, multiple isoforms of GPAT are expressed and differentially regulated in epidermis/keratinocytes.

In vitro culture of Keratinocytes from human umbilical cord blood mesenchymal stem cells: the Saigonese culture

There have been many attempts to acquire and culture human keratinocytes for clinical purposes including from keratotome slices in media with fetal calf serum (FCS) or pituitary extract (PE), from skin specimens in media with feeder layers, from suction blister epidermal roofs' in serum-free culture and from human umbilical cord blood (hUCB) mesenchymal stem cells (MSCs) in media with skin feeder layers. Conversely this study was designed to investigate whether keratinocytes could be obtained directly from hUCB MSCs in vitro. It is widely established that mesenchymal stem cells from human umbilical cord blood have multipotent capacity and the ability to differentiate into disparate cell lineages hUCB MSCs were directly induced to differentiate into keratinocytes by using a specific medium composed of primary culture medium (PCM) and serum free medium (SFM) in a ratio 1:9 for a period of 7days and tested by immunostain p63 and K1-K10. Cells thus cultured were positive in both tests, confirming the possibility to directly obtain keratinocytes from MSCs hUCB in vitro.

A Thyroid Hormone Deiodinase Inhibitor Can Decrease Cutaneous Cell Proliferation In Vitro

Although cutaneous manifestations associated with thyroid dysfunction are classic, the potential for thyroid hormone or its antagonists to treat dermatological disease has not been explored with rigor. The predominant circulating thyroid hormone is the pro-hormone, thyroxine (T(4)). Skin, like many tissues, expresses thyroid hormone deiodinases to convert T(4) to the active thyroid hormone, triiodothyronine (T(3)). Previously, we determined that T(3) is necessary for optimal growth of keratinocytes and fibroblasts. The first hypothesis of this experiment was that the deiodinase inhibitor iopanoic acid (IOP) could inhibit cutaneous cell proliferation. The second hypothesis of this experiment was that the action of IOP could be attributed to its inhibition of conversion of T(4) to T(3). Although IOP is known to inhibit T(4) to T(3) conversion, the inhibition of cutaneous cell proliferation by IOP might conceivably result from other properties of IOP. In separate experiments, primary culture human keratinocytes and dermal fibroblasts were incubated overnight with IOP. Proliferation was inhibited in a dose-dependent manner in both cell lines. Overnight incubation with T(3) restored the proliferation but overnight incubation with T(4) did not. The study is the first to demonstrate that IOP inhibits cutaneous cell proliferation and that supplemental T(3) is sufficient to correct at least part of the inhibition caused by IOP. The data suggest that IOP decreases cutaneous cell proliferation by inhibition of intracellular T(4) to T(3) conversion. The data provide indirect evidence of the presence of type 1 or type 2 iodothyronine deiodinase activity in skin cells. The data support our previous hypothesis that T(3) is necessary for normal cutaneous proliferation.

Decreased Ceramide Transport Protein (CERT) Function Alters Sphingomyelin Production following UVB Irradiation

Increased cellular ceramide accounts in part for UVB irradiation-induced apoptosis in cultured human keratinocytes with concurrent increased glucosylceramide but not sphingomyelin generation in these cells. Given that conversion of ceramide to non-apoptotic metabolites such as sphingomyelin and glucosylceramide protects cells from ceramide-induced apoptosis, we hypothesized that failed up-regulation of sphingomyelin generation contributes to ceramide accumulation following UVB irradiation. Because both sphingomyelin synthase and glucosylceramide synthase activities were significantly decreased in UVB-irradiated keratinocytes, we investigated whether alteration(s) in the function of ceramide transport protein (or CERT) required for sphingomyelin synthesis occur(s) in UVB-irradiated cells. Fluorescently labeled N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C(5)-DMB-ceramide) relocation to the Golgi was diminished after irradiation, consistent with decreased CERT function, whereas the CERT inhibitor N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (1R,3R isomer) (HPA-12) produced an equivalent effect. UVB irradiation also induced the rapid formation of a stable CERT homotrimer complex in keratinocytes as determined by Western immunoblot and mass spectrometry analyses, a finding replicated in HeLa, HEK293T, and HaCaT cells and in murine epidermis. Ceramide binding activity was decreased in recombinant CERT proteins containing the UVB-induced homotrimer. The middle region domain of the CERT protein was required for the homotrimer formation, whereas neither the pleckstrin homology (Golgi-binding) nor the START (ceramide-binding) domains were involved. Finally like UVB-treated keratinocytes, HPA-12 blockade of CERT function increased keratinocyte apoptosis, decreased sphingomyelin synthesis, and led to accumulation of ceramide. Thus, UVB-induced CERT homotrimer formation accounts, at least in part, for apoptosis and failed up-regulation of sphingomyelin synthesis following UVB irradiation, revealing that inactive CERT can attenuate a key metabolic protective mechanism against ceramide-induced apoptosis in keratinocytes.

On the effects of topical synthetic pseudoceramides: Comparison of possible keratinocyte toxicities provoked by the pseudoceramides, PC104 and BIO391, and natural ceramides

Ceramides (Cer) in the stratum corneum are essential for epidermal permeability barrier function. Thus, topical Cer replacement therapy has been employed to improve barrier function in clinical situations associated with Cer deficiency, e.g., atopic dermatitis. Because of the disadvantages of both natural- and skin identical-Cer (central nervous system origins and cost, respectively), synthetic chemical mimics, or pseudoceramides (pseudo-Cer), have been utilized as Cer substitutes. Whereas increased levels of intracellular Cer trigger cell growth inhibition and apoptosis, Cer levels are maintained by metabolic/catabolic pathways protecting cells from Cer-induced apoptosis. However, since the metabolic fates of each pseudo-Cer remain unknown, their widespread deployment in topical agents has raised concern about potential toxicities. We compared the effects of two chemically unrelated commercially available pseudo-Cer to exogenous cell-permeant (C2)- or natural (C18)-Cer on cell growth and apoptosis thresholds in cultured human keratinocytes (CHK). Cell growth and cell toxicity of CHK exposed to either C2-Cer or pseudo-Cer were assessed by MTT and lactate dehydrogenase release assays. Mitochondrial membrane potential, an indicator of apoptosis, was measured using membrane permeabilized semi-intact keratinocytes exposed C2-Cer, natural-Cer or pseudo-Cer. While the cell-permeant-Cer inhibits keratinocyte growth and increases cell toxicity, neither of the pseudo-Cer showed these effects. Decreased mitochondrial membrane potential occurred in CHK incubated with cell-permeant- and natural-Cer, but not pseudo-Cer. Taken together with preclinical safety studies of these pseudo-Cer and their widespread use over the counter without evidence of toxicity, these studies provide further assurance about the safety of these pseudo-Cer for topical use.

Regulation of ABCA1 expression in human keratinocytes and murine epidermis

Keratinocytes require abundant cholesterol for cutaneous permeability barrier function; hence, the regulation of cholesterol homeostasis is of great importance. ABCA1 is a membrane transporter responsible for cholesterol efflux and plays a pivotal role in regulating cellular cholesterol levels. We demonstrate that ABCA1 is expressed in cultured human keratinocytes (CHKs) and murine epidermis. Liver X receptor (LXR) activation markedly stimulates ABCA1 mRNA and protein levels in CHKs and mouse epidermis. In addition to LXR, activators of peroxisome proliferator-activated receptor (PPAR)alpha, PPARbeta/delta, and retinoid X receptor (RXR), but neither PPARgamma nor retinoic acid receptor, also increase ABCA1 expression in CHKs. Increases in cholesterol supply induced by LDL or mevalonate stimulate ABCA1 expression, whereas inhibiting cholesterol synthesis with statins or cholesterol sulfate decreases ABCA1 expression in CHKs. After acute permeability barrier disruption by either tape-stripping or acetone treatment, ABCA1 expression declines, and this attenuates cellular cholesterol efflux, making more cholesterol available for regeneration of the barrier. In addition, during fetal epidermal development, ABCA1 expression decreases at days 18-22 of gestation (term = 22 days), leaving more cholesterol available during the critical period of barrier formation. Together, our results show that ABCA1 is expressed in keratinocytes, where it is negatively regulated by a decrease in cellular cholesterol levels or altered permeability barrier requirements and positively regulated by activators of LXR, PPARs, and RXR or increases in cellular cholesterol levels.

LXR and PPAR activators stimulate cholesterol sulfotransferase type 2 isoform 1b in human keratinocytes

Liver X receptors (LXRs) and peroxisome proliferator-activated receptors (PPARs) are potent regulators of keratinocyte proliferation, differentiation, and epidermal permeability barrier homeostasis. Cholesterol sulfotransferase type 2B isoform 1b (SULT2B1b) is a key enzyme in the synthesis of cholesterol sulfate (CS), a critical regulator of keratinocyte differentiation and desquamation, as well as a mediator of barrier homeostasis. In this study, we assessed the effect of activators of LXR, PPARalpha, PPARbeta/delta, and PPARgamma on SULT2B1b gene expression and enzyme activity in cultured human keratinocytes (CHKs). Our results demonstrate that PPAR and LXR activators increase SULT2B1b mRNA levels, with the most dramatic effect (a 26-fold increase) induced by the PPARgamma activator ciglitazone. Ciglitazone upregulates SULT2B1b mRNA in a dose- and time-dependent manner. Moreover, the stimulation of SULT2B1b gene expression by LXR and PPAR activators occurs in both undifferentiated and differentiated CHKs. The upregulation of SULT2B1b mRNA by ciglitazone appears to occur at a transcriptional level, because the degradation of SULT2B1b is not accelerated by ciglitazone. In addition, cycloheximide almost completely blocks the ciglitazone-induced increase in SULT2B1b mRNA, suggesting that the transcription of SULTB1b mRNA is dependent on new protein synthesis. Finally, LXR and PPAR activators also increased the activity of cholesterol sulfotransferase. Thus, LXR and PPAR activators regulate the expression of SULT2B1b, the key enzyme in the synthesis of CS, which is a potent regulator of epidermal differentiation and corneocyte desquamation.

Dimethyl sulfoxide and oxidative stress on cultures of human keratinocytes

The aim of the present study was to examine the protective action of the antioxidant dimethyl sulfoxide (DMSO) against the oxidative stress on keratinocyte cultures caused by glucose deprivation and hypoxia, using the concentration of malonyl dialdehyde existing in the cell culture as an indicator of the oxidative stress level. EIGHTY FLASKS WITH CULTURED HUMAN KERATINOCYTES IN A CONFLUENT LAYER WERE DIVIDED INTO EIGHT GROUPS, INCLUDING THE FOLLOWING: culture medium with and without glucose, culture medium with and without the addition of DMSO, culture medium subjected and not subjected to hypoxia, and culture medium with a combination of these factors. The statistical analysis of the results showed that DMSO proved to be an effective agent against the oxidative stress on cultures of keratinocytes under the experimental conditions studied.

Glucosylceramide synthesis and synthase expression protect against ceramide-induced stress

Ceramides (Cers), critical for epidermal barrier function, also can inhibit keratinocyte proliferation, while glucosylceramides (GlcCers) exert pro-mitogenic effects. Since alterations in Cer-to-GlcCer ratios appear to modulate cellular growth versus apoptosis, we assessed whether keratinocytes up-regulate GlcCer synthesis as a protective mechanism against Cer-induced stress. Exogenous sphingomyelinase (SMase) treatment of cultured human keratinocytes (CHK) initially decreased proliferation and cellular sphingomyelin (50-60% decrease; P < 0.001), and increased Cer levels (6.1- to 6.8-fold; P < 0.001). Proliferation recovered to normal rates by 24 h, in parallel with increased cellular GlcCer. Both GlcCer synthesis and GlcCer synthase activity increased significantly by 8 h following SMase (8.2- and 2.4-fold, respectively; P < 0.01 each vs. control), attributed to antecedent increases in GlcCer synthase mRNA and protein expression. Further evidence that GlcCer production is responsible for normalized CHK proliferation includes: a) attenuation of SMase-induced inhibition of proliferation by exogenous GlcCer; and b) enhancement of the SMase effect in cells cotreated with the GlcCer synthase inhibitor, PDMP (D-threo-1-phenyl-2(decanoylamino)-3-morpholino-1-propanol). Finally, although proliferation in immortalized, nontransformed keratinocytes (HaCaT) also was inhibited by SMase, HaCaT cells that overexpress GlcCer synthase were resistant to this effect. Thus, SMase-induced stress initiates a response in keratinocytes that includes upregulation of GlcCer synthesis which may protect against the deleterious effects of excess Cer.

Mechanistic Studies to Evaluate the Enhanced Antiproliferation of Human Keratinocytes by 1α,25-Dihydroxyvitamin D3–3-bromoacetate, a Covalent Modifier of Vitamin D Receptor, Compared to 1α,25-Dihydroxyvitamin D3

1α,25-Dihydroxyvitamin D3–3-bromoacetate (1,25(OH)2D3-3-BE), an affinity labeling analog of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), displayed stronger antiproliferative activities than 1,25(OH)2D3 at 10−10–10−6 M dose levels in cultured human keratinocytes (CHK). Additionally, preincubation of the cells with 10−6 M 1,25(OH)2D3, followed by treatment with various doses of 1,25(OH)2D3-3-BE, resulted in a significantly stronger antiproliferative activity by the mixture than individual reagents at every dose level. To search for a mechanism of this observation, we determined that [14C]1,25(OH)2D3-3-BE covalently labeled human recombinant 1α,25-dihydroxyvitamin D3 receptor (reVDR) swiftly (<1 min) with a 1:1 stoichiometry and induced conformational changes (in VDR) that are different from 1,25(OH)2D3, by limited tryptic digestion. Furthermore, a protein band, corresponding to reVDR, was specifically labeled by [14C]1,25(OH)2D3-3-BE in CHK extract, indicating that VDR is the main target of [14C]1,25(OH)2D3-3-BE. The above-mentioned observations suggest that a rapid covalent labeling of VDR in CHK might alter the interaction between the holo-VDR and 1,25(OH)2D3-controlled genes. Furthermore, we observed that 1,25(OH)2D3-3-BE significantly decreased the binding of VDR to human osteocalcin vitamin D responsive element (hOCVDRE), as well as the dissociation rate of VDR from hOCVDRE, compared with 1,25(OH)2D3 in COS-1 cells, transiently transfected with a VDR construct. Additionally, 1,25(OH)2D3-3-BE was found to be more potent in inducing 1α,25-dihydroxyvitamin D3–24-hydroxylase (24-OHase) promoter activity and mRNA expression in keratinocytes. The accumulation of 24-OHase message was also prolonged by the analog. Collectively these results indicated that rapid covalent labeling of VDR in keratinocytes (by 1,25(OH)2D3-3-BE) might result in the conversion of apo-VDR to a holo-form, with a conformation that is different from that of the 1,25(OH)2D3–VDR complex. This resulted in an enhanced stability of the 1,25(OH)2D3-3-BE/VDR–VDRE complex and contributed to the amplified antiproliferative effect of 1,25(OH)2D3-3-BE in keratinocytes.

UVB irradiation up-regulates serine palmitoyltransferase in cultured human keratinocytes

The enzyme serine palmitoyltransferase (SPT; EC 2.3.1.50), which catalyzes the first committed and rate-limiting step in sphingolipid synthesis, is up-regulated in the epidermis as part of the homeostatic repair in response to permeability barrier perturbation. Moreover, UVB exposure, which also perturbs the barrier, up-regulates sphingolipid synthesis, but the basis for this increase is not known. The recent isolation of cDNAs for SPT (i.e., LCB1 and LCB2) allow molecular regulation studies to be performed. Therefore, we determined whether UVB exposure alters mRNA, protein, or activity levels for SPT in cultured human keratinocytes (CHKs) as a mechanism for regulation of epidermal sphingolipid synthesis. In CHK, transcripts for both LCB1 (3.0 kb) and LCB2 (2.3 kb) are evident by Northern blot analysis, and UVB exposure (23 mJ/cm2) induces a delayed 1.8 to 3.3-fold increase in LCB2 mRNA levels (P < 0.01) 48 h after treatment versus non-irradiated control cells. In contrast, neither LCB1 nor a second LCB2 transcript (8.0 kb) changed significantly. Likewise, Lcb2 protein levels (by Western blot analysis), as well as SPT activity, increase in parallel with the increased LCB2 mRNA. Finally, incorporation of [14C]-acetate into sphingolipids was increased significantly 48 h after UVB treatment. Together, these results demonstrate that CHKs respond to UVB by increasing sphingolipid synthesis, primarily through increases in both LCB2 mRNA and protein levels, leading to increased SPT activity. These results demonstrate one mechanism (UVB) whereby SPT is regulated at the molecular level, and suggest further that epidermis up-regulates sphingolipid synthesis at both the mRNA and protein levels in response to UVB.—Farrell, A. M., Y. Uchida, M. M. Nagiec, I. R. Harris, R. C. Dickson, P. M. Elias, and W. M. Holleran. UVB irradiation up-regulates serine palmitoyltransferase in cultured human keratinocytes. J. Lipid Res. 1998. 39: 2031–2038.

Expression of 25-Hydroxyvitamin D3-24-Hydroxylase mRNA in Cultured Human Keratinocytes

It is well documented that 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25[OH]2D3), the most active vitamin D metabolite, inhibits epidermal keratinocyte proliferation and promotes differentiation. 1 alpha,25(OH)2D3 can be produced in keratinocytes from 25-hydroxyvitamin D3 by the enzyme 25-hydroxyvitamin D3-1 alpha-hydroxylase (1-OHase). Hydroxylation of 1 alpha,25(OH)2D3 by 25-hydroxyvitamin D3-24-hydroxylase (24-OHase), the first step in the catabolic pathway of 1 alpha,25(OH)2D3 could significantly reduce the intracellular concentration of 1 alpha,25(OH)2D3. Therefore, the expression of 24-OHase could have a critical regulatory role in 1 alpha,25(OH)2D3-dependent gene expression. As a first step to examine this possibility, the steady state level of 24-OHase mRNA in cultured human keratinocytes (CHK) was investigated. 24-OHase mRNA was not detected in control CHK. 1 alpha,25(OH)2D3 caused a dose- and time-dependent increase in 24-OHase mRNA level. The highest accumulation of 24-OHase mRNA was observed in CHK treated with 0.1-1 microM 1 alpha,25(OH)2D3. The level of 24-OHase mRNA reached a plateau 12-24 hr after 1 alpha,25(OH)2D3 treatment. 1 beta,25-dihydroxyvitamin D3, the stereoisomer of 1 alpha,25(OH)2D3, failed to induce 24-OHase mRNA expression significantly. In addition to 24-OHase mRNA, a 1.0-kb mRNA hybridized strongly with both rat and human 24-OHase cDNA probes. The origin of this 1.0-kb message is unknown at present, however, it was regulated by 1 alpha,25(OH)2D3. These results demonstrate that 1 alpha,25(OH)2D3 up-regulates the expression of 24-OHase mRNA, and this may be an important first step in the initiation of catabolism of 1 alpha,25(OH)2D3 in human keratinocytes.

Keratinocyte grafts for wound healing

Attempts by early investigators to culture human keratinocytes in vitro were hampered by fibroblast overgrowth in culture. In addition, under the culture conditions at the time, the keratinocytes rapidly differentiated and did not actively propagate.1 In the 1960s tissue expiant cultures were used in attempts to obtain improved epidermal expansion. In this technique, a skin sample was placed with the dermal surface in contact with the culture dish to allow attachment and then incubated in culture medium. The epidermis grew down the side of the explant, attached to the dish, and then migrated outward to produce epithelial layers that could be used as allografts in animal models2; however, proliferation of epidermal cells using this method was insufficient to produce the large amounts of epidermis required for grafting.3–5 In 1975 Rheinwald and Green described a significant advance in keratinocyte culture techniques that allowed serial subcultivation of human keratinocytes. Whole skin was trypsinized to produced a single-cell suspension of keratinocytes. These were then cultivated on a feeder layer of lethally irradiated 3T3 fibroblasts. The technique has been refined and modified so that it is now possible, starting with a skin sample of 1 cm2, to expand the amount of cultured epithelium 10,000-fold to a size comparable to that of the adult body surface. Using the enzyme dispase, the epidermal sheet can be detached from the culture dish (Fig 1). A nonadherent dressing or vaseline gauze is then placed on the outer surface as a backing to facilitate handling of the graft6–10 (Fig 2).An important advance in this methodology was the introduction of epidermal growth factor, which was a potent stimulator of keratinocyte yield in culture.10 In addition, many new keratinocyte culture techniques using a chemically more defined medium have been described by multiple groups.11–18 From grafting experiments on nude mice, it was found that cultured epidermal cells regenerate a fully differentiated epidermis with normal architecture and a relatively complete pattern of keratinization including synthesis of high-molecular-weight keratins.19

Cultured human keratinocytes cannot metabolize vitamin D 3 to 25-hydroxyvitamin D 3

The metabolism of [ 3H] vitamin D 3 was studied in cultured human keratinocytes (CHK). Intact CHK were incubated for 1, 6, 12, 24 and 48 h with [ 3H] vitamin D 3 and the lipid soluble fractions from the media and cells were extracted by high-performance liquid chromatography (HPLC). Vitamin D 3 and its metabolites, 25-OH-D 3, 24,25(OH) 2D 3 were added to the extracts, as markers, prior to HPLC, HPLC analysis of the lipid extracts did not reveal any monohydroxylated metabolites. CHK incubated for one hour with [ 3H]25-OH-D 3 showed a 10±4% conversion to [ 3H]1,25,(OH) 2D 3 whereas no conversion to [ 3H]1,25(OH) 2D 3 was observed in control CHKs that were boiled prior to incubation with [ 3H]25-OH-D 3. These findings suggest that cultured neonatal keratinocytes are incapable of metabolizing vitamin D 3 to 25-OH-D 3.