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J. Biol. Chem., Vol. 277, Issue 4, 2916-2922, January 25, 2002

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Scavenger Receptor Class B Type I Is Expressed in Cultured Keratinocytes and Epidermis

REGULATION IN RESPONSE TO CHANGES IN CHOLESTEROL HOMEOSTASIS AND BARRIER REQUIREMENTS^*

Hiroki Tsuruoka^§¶, Weerapan Khovidhunkit^**, Barbara E. Brown^§, Joachim W. Fluhr^§, Peter M. Elias^§, and Kenneth R. Feingold^§**

From the  Dermatology and  Medical (Metabolism) Services, Department of Veterans Affairs Medical Center, San Francisco, California 94121, the Departments of ^§ Dermatology and ^** Medicine, University of California, San Francisco School of Medicine, San Francisco, California 94143, and ^¶ R & D Department of Dermatological Science, POLA Laboratories, POLA Chemical Industries Inc., 560 Kashio-cho, Totsuka-ku, Yokohama 244-0812 Japan

Received for publication, July 10, 2001, and in revised form, November 12, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cholesterol is a key lipid in the stratum corneum, where it is critical for permeability barrier homeostasis. The epidermis is an active site of cholesterol synthesis, but inhibition of epidermal cholesterol synthesis with topically applied statins only modestly affects epidermal permeability barrier function, suggesting a possible compensatory role for extraepidermal cholesterol. Scavenger receptor class B type I (SR-BI) is a recently described cell surface receptor for high density lipoproteins (HDL) that mediates the selective uptake of cholesterol esters from circulating HDL. In the present study, we demonstrate that SR-BI is present in cultured human keratinocytes and that calcium-induced differentiation markedly decreases SR-BI levels. Additionally, the cell association of [³H]cholesterol-labeled HDL decreased in differentiated versus undifferentiated keratinocytes. Furthermore, the inhibition of cholesterol synthesis with simvastatin resulted in a 3-4-fold increase in both SR-BI mRNA and protein levels, whereas conversely, addition of 25-hydroxycholesterol suppressed SR-BI levels by approximately 50%. SR-BI mRNA is also expressed in murine epidermis, increasing by 50% in parallel with cholesterol requirements following acute barrier disruption. Because the increase is completely blocked by occlusion with a vapor-impermeable membrane, changes in epidermal SR-BI expression are regulated specifically by barrier requirements. Lastly, using immunofluorescence we demonstrated that SR-BI is present in human epidermis predominantly in the basal layer and increases following barrier disruption. In summary, the present study demonstrates first that SR-BI is expressed in keratinocytes and regulated by cellular cholesterol requirements, suggesting that it plays a role in keratinocyte cholesterol homeostasis. Second, the increase in SR-BI following barrier disruption suggests that SR-BI expression increases to facilitate cholesterol uptake leading to barrier restoration.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A major function of the skin is to form a cutaneous permeability barrier to prevent the excessive loss of water and solutes from the body. Extracellular lamellar membranes in the stratum corneum that contain primarily ceramides, fatty acids, and cholesterol provide this permeability barrier (1). These lipid lamellar membranes are delivered to the stratum corneum via the secretion of lamellar body contents from highly differentiated keratinocytes in the outermost stratum granulosum layer of the epidermis (1). Both the formation and the secretion of lamellar bodies are regulated specifically by barrier requirements; disruption of the permeability barrier results in: (a) rapid delivery of profound lamellar body contents to the stratum corneum-stratum granulosum interface (2), followed by (b) the accelerated formation and secretion of nascent lamellar bodies (2). The specific link to the barrier is shown by the fact that artificial barrier restoration by occlusion with an impermeable membrane prevents both the formation and secretion of nascent lamellar bodies (2). The formation of lamellar bodies requires the generation of abundant lipid precursors; accordingly, inhibition of cholesterol, ceramide, or fatty acid synthesis inhibits lamellar body formation and secretion (3-5).

Even under basal, unperturbed conditions, the epidermis is a very active site of cholesterol synthesis (6, 7). Yet disruption of the permeability barrier results in a further increase in epidermal cholesterol synthesis (8, 9), which is accompanied by increases in the activities, protein, and mRNA levels of 3-hydroxy-methylglutaryl CoA (HMG-CoA)¹ reductase (9, 10). Likewise, the expression of other key enzymes in the cholesterol synthetic pathway, including HMG-CoA synthase, farnesyl pyrophosphate synthase, and squalene synthase, also increase with permeability barrier disruption (11). Occlusion with a vapor-impermeable membrane prevents the increase in cholesterol synthesis following barrier disruption (6, 7), indicating that changes in transepidermal water movement regulate epidermal cholesterol synthesis. Furthermore, topical applications of HMG-CoA reductase inhibitors block epidermal cholesterol synthesis and delay barrier recovery following barrier disruption (3, 12), indicating that epidermal cholesterol synthesis is critical for normal cutaneous permeability barrier homeostasis. Yet despite a marked inhibition of epidermal cholesterol synthesis, some lamellar bodies continue to be formed, and recovery of permeability barrier function proceeds in the face of inhibitor blockade, albeit at a modestly delayed rate (3). These results suggest that cholesterol could be delivered to the epidermis from extracutaneous sites following barrier disruption, providing lipid precursors and allowing replenishment of lamellar bodies leading to barrier recovery.

Lipid uptake into cells is mediated by receptors on the plasma membrane. The LDL receptor, which is capable of binding lipoproteins that contain apolipoprotein B or apolipoprotein E, is present on keratinocytes (13, 14), but LDL receptor density declines above the basal layer (13, 15). Thus, the LDL receptor is positioned to be involved in the uptake of LDL from the circulation into epidermis. Because LDL receptor protein and mRNA levels increase following barrier disruption (10), it is likely that LDL receptor-mediated lipid uptake plays a role in permeability barrier homeostasis.

Recently, the scavenger receptor class B type I (SR-BI) receptor was identified and shown to mediate the selective uptake of cholesterol from lipoprotein particles by a mechanism that requires neither endocytosis nor degradation of the entire particle (16-19). SR-BI is abundantly expressed both in the liver and in steroidogenic tissues that require cholesterol for synthetic processes (17, 20, 21). In the adrenal gland and ovary, SR-BI expression is up-regulated when the requirements for cholesterol for steroid synthesis increase (20, 22). Moreover, SR-BI receptor knockout mice demonstrate a marked decrease in adrenal cholesterol stores (23), further demonstrating the importance of this receptor as a pathway for cholesterol uptake. Thus, SR-BI is a key lipoprotein receptor of cholesterol homeostasis in certain tissues.

Because of the requirement for large amounts of cholesterol by the epidermis for the formation of lamellar bodies and the cutaneous permeability barrier, we hypothesized that keratinocytes would express SR-BI. In the present study, we demonstrate that: (a) SR-BI is present in cultured human keratinocytes and in both murine and human epidermis, where it localizes mostly to the basal layer, (b) SR-BI expression decreases with keratinocyte differentiation and cholesterol excess but increases with sterol depletion, and (c) barrier disruption up-regulates SR-BI expression in a manner that is blocked by artificial barrier restoration. Together, these results suggest that SR-BI may be important for the restoration of extracellular cholesterol needed to maintain permeability barrier homeostasis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- [-³²P]dCTP (3000 µCi/ml) was purchased from ICN Pharmaceuticals Inc. (Costa Mesa, CA). The Multiprime DNA labeling system was purchased from Amersham Biosciences, Inc. SR-BI cDNA probe was generated by reverse transcriptase-PCR from the mouse liver (oligonucleotide primers: upper, 5'-ATGCAGGTCCATGAAGCTGAC-3', and lower, 5'-CTATAGCTTGGCTTCTTGCAGC-3'). 25-OH cholesterol and simvastatin were purchased from Sigma and Calbiochem (San Diego, CA), respectively.

Keratinocyte Culture-- Human foreskin keratinocytes, second passage, were maintained in 0.07 mM Ca²⁺ keratinocyte growth medium (KGM; Clonetics, San Diego, CA). To determine the effect of differentiation, culture media were changed to KGM containing either 0.03 or 1.2 mM Ca²⁺ when the cells reached 80% confluence. These cells were maintained for 7 days changing the media once every 2 days. In some experiments, human foreskin keratinocytes, second passage, were grown to 80-90% confluence in 0.07 mM Ca²⁺ KGM. The cells were then treated with 25-OH cholesterol (12.5 µM), simvastatin (10 µM), or vehicle (ethanol) for 24 h for Northern blot and 48 h for Western blot analysis.

Isolation of Poly(A)⁺ RNA from Cultured Keratinocytes-- Poly(A)⁺ RNA was isolated as described previously (24). Briefly, human keratinocytes were lysed in 0.5 M NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA, 1% sodium dodecyl sulfate, and 200 µg of proteinase K/ml. After the incubation for 1 h at 37 °C, oligo(dT)-cellulose (Amersham Biosciences, Inc.) was added and incubated for 1 h at room temperature. After the oligo(dT)-cellulose was washed, the poly(A)⁺ RNA was eluted with diethyl pyrocarbonate-treated water and precipitated with ethanol.

Barrier Disruption and Isolation of Poly(A)⁺ RNA from Mouse Epidermis-- Male hairless mice (Charles River Laboratories, Wilmington, MA), 8-10 weeks old, were treated either with sequential applications of cellophane tape or with acetone until the transepidermal water loss reached 6-10 mg/cm²/h measured with an electrolytic moisture analyzer (Meeco, Inc., Warrington, PA) as described previously (6). Occlusion was performed by wrapping mice with one finger of a Latex glove immediately after barrier disruption as described previously (25). To separate whole epidermis, the skin was incubated in 10 mM EDTA in calcium and magnesium-free PBS, pH 7.4, for 30 min at 37 °C. The epidermis was then scraped off the dermis with a scalpel. Total RNA was prepared by the guanidinium thiocyanate method as described previously (26). Total RNA was purified, and poly(A)⁺ was isolated with oligo(dT)-cellulose and precipitated with ethanol as described above.

Northern Blot Analysis-- The concentration of RNA was determined spectrophotometrically. Equal amounts of poly(A)⁺ (8-10 µg) were separated by electrophoresis through 1% agarose-formaldehyde gels. The RNA was blotted onto nylon membranes (Schleicher & Schuell) after electrophoresis. [³²P]dCTP-labeled cDNA probes were prepared by the Multiprime DNA labeling system (Amersham Biosciences, Inc.) following the instructions. Blots were prehybridized with ULTRAhyb^TM (Ambion, Austin, TX) for 1 h, followed by hybridization with ³²P-labeled probe for 16 h at 65 °C. After hybridization, the blots were washed with a solution of 0.2% SSC, 0.1% SDS for 15 min at room temperature, followed by a 15-min wash at 65 °C. Autoradiography was performed at 70 °C. The relative intensities of the bands were determined by densitometry GS 710 (Bio-Rad). The blots were probed with either -actin or cyclophilin to normalize data.

Western Blot Analysis-- Cultured keratinocytes were homogenized by sonication in 20 mM Tris-HCl, pH 7.4, containing a protease inhibitor mixture (Roche Molecular Biochemicals). The homogenate was centrifuged at 3,000 × g for 5 min at 4 °C to remove debris, and then the supernatant was collected and centrifuged at 12,000 × g for 20 min at 4 °C. The pellet was rehomogenized in the same buffer as above plus 150 mM NaCl at 4 °C. Protein levels were determined by BCA protein assay kit (Pierce). Equal amounts (50 µg) of protein were electrophoresed on 8.5% polyacrylamide gels, and the separated proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad). Polyclonal anti-SR-BI antibody (1:1000; Novus Biochemicals Inc., Littleton, CO) was used to determine SR-BI protein levels, followed by alkaline phosphatase-conjugated anti-rabbit IgG secondary antibody (1:2000; Amersham Biosciences, Inc.) and chemiluminescent detection (Amersham Biosciences, Inc.). The relative intensities of the bands were determined by densitometry GS 710 (Bio-Rad).

Cholesterol Synthesis-- Cholesterol synthesis was determined as described previously (27). Briefly, human keratinocytes were grown in 0.07 mM calcium KGM to 90% confluence and then treated with either ethanol or 10 µM simvastatin for 24 h. [¹⁴C]Acetate (20 µCi/ml; American Radiolabeled Chemicals, St. Louis, MO) and 1 mM cold acetate (Sigma) were added to the cultured keratinocytes for the last 3 h before harvest. The lipids were extracted with petroleum ether after saponification and separated by thin layer chromatography. The band corresponding to standard cholesterol was scraped from the plate, and the incorporation of [¹⁴C]acetate into cholesterol was determined as amount of cholesterol synthesis. The values were normalized by protein amount and expressed as percentages of control.

Immunofluorescence Microscopy-- Cultured human keratinocytes maintained either in low calcium (0.03 mM) or high calcium (1.2 mM) KGM for 7 days were fixed in 4% paraformaldehyde for 10 min at room temperature. After 1 h of incubation with blocking buffer (0.1% fish gelatin, 0.8% bovine serum albumin, and 0.01% Tween 80 in PBS), the slides were treated with polyclonal anti-SR-BI antibody (1:200; Novus Biochemicals Inc.) overnight at 4 °C, followed by fluorescein-conjugated anti-rabbit Ig G antibody (1:200; DAKO, Carpinteria, CA). Counterstaining for nuclei was carried out with propidium iodide (Sigma). After washing, the coverslips were mounted, and the samples were viewed using a confocal microscope (Zeiss, Heidelberg, Germany).

Punch biopsies were taken from the forearms of a healthy volunteer. The basal transepidermal water loss was measured with a Tewameter TM210 (Courage + Khazaka, Köln, Germany) on both arms, and then one arm was tape stripped until an ~8-fold increase in transepidermal water loss. The equivalent site on the other arm served as a basal state. Transepidermal water loss was measured again 3 h after tape stripping, and biopsies were carried out. Frozen sections were fixed in cold acetone for 10 min at 20 °C and processed as follows. After 1 h of incubation with blocking buffer (0.1% fish gelatin, 2% bovine serum albumin, and 0.01% Tween 80 in PBS), the slides were treated with purified polyclonal anti-SR-BI antibody (1:100; Novus Biochemicals) overnight at 4 °C, followed by fluorescein-conjugated anti-rabbit IgG antibody (1:100; Vector Laboratories, Burlingame, CA) for 2 h at room temperature. The sections were counterstained with propidium iodide (Sigma). After washing, the coverslips were mounted, and the samples were viewed using a confocal microscope. The sections, when the anti-SR-BI antibody was omitted, served as a negative control.

Lipoprotein Preparation-- Human high density lipoprotein subfraction 3 was isolated from the fresh plasma of a healthy volunteer by density gradient ultracentrifugation method as described by Redgrave et al. (28). Isolated HDL (see Fig. 4A) was dialyzed against 1 mM EDTA-PBS, and then HDL was labeled with [³H]cholesteryl oleolyl ether (Amersham Biosciences, Inc.) as follows. Purified HDL (1.5 mg of protein) was mixed with [³H]cholesteryl oleolyl ether (100 µCi) and lipoprotein-deficient plasma on a glass microfiber in a scintillation vial. The mixture was incubated at 37 °C for 18 h shaking gently. Radiolabeled HDL was isolated by density gradient ultracentrifugation and dialyzed against 1 mM EDTA-PBS (specific activity, 7.5 × 10⁴ dpm/µg CE was obtained). The content of total cholesterol and free cholesterol was measured by a cholesterol CII kit (Wako Chemicals) and a free cholesterol C kit (Wako Chemicals), respectively. The protein levels were determined by BCA protein assay kit (Pierce).

HDL Cell Association-- 50 µg/ml protein of labeled HDL in the presence or absence of a 25-fold excess of unlabeled HDL was added to keratinocytes cultured in either 0.03 or 1.2 mM Ca²⁺ KGM for 7 days. After incubation at 37 °C for 3 h, the medium was removed, and the cells were washed three times with 0.1% bovine serum albumin in PBS (pH 7.4) and a fourth time with PBS alone. The cells were lysed in PBS by sonication, and the cell association of HDL was determined by counting tritium radioactivity in the cell homogenate.

Statistics-- Statistical analyses were performed using a two-tailed Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

SR-BI Is Expressed in Cultured Human Keratinocytes and Down-regulated by Calcium-induced Differentiation-- We first assessed whether SR-BI is present in keratinocytes and regulated by changes in extracellular calcium. As shown in Fig. 1, a single band corresponding to SR-BI mRNA is detected by Northern blotting of mRNA from cultured human keratinocytes, grown under proliferative conditions (0.03 mM calcium). The transcript size of SR-BI mRNA under these conditions is similar to that reported in other human tissues (~2.8 kb) (21). We and others have shown that cultured human keratinocytes remain in a proliferative state when grown in low calcium (0.03-0.07 mM), whereas exposure to higher calcium concentrations (>0.1 mM) inhibits proliferation inducing several keratinocyte-specific differentiation-linked proteins (29-33). In contrast, exposure to high calcium (1.2 mM) leads to a decrease in SR-BI mRNA levels (~70%) (Fig. 1).

View larger version (31K): [in this window] [in a new window]   Fig. 1.   SR-BI mRNA levels in cultured human keratinocytes decrease with differentiation. Human keratinocytes were grown either in low calcium (0.03 mM) or high calcium (1.2 mM) KGM for 7 days. Northern blots were probed for SR-BI mRNA as described under "Experimental Procedures." mRNA values were normalized to those of corresponding -actin measured on the same blot and expressed relative to a control value of one. A representative blot is shown here. The values are the means ± S.E. n = 6 in each group. *, p < 0.01.

To determine whether the calcium-induced decrease in SR-BI mRNA levels leads to a reduction in SR-BI protein levels, we next quantitated SR-BI protein levels in Western blots from human keratinocytes grown in either 0.03 or 1.2 mM calcium for 7 days. As shown in Fig. 2, a band was detected at ~80 kDa, which is similar to the molecular mass of SR-BI protein reported in other human tissues (21). Moreover, as observed with mRNA measurement, SR-BI protein levels declined significantly in 1.2 mM calcium (~70%). In addition, immunofluorescence analysis visualized a strong green signal for SR-BI that was localized to the plasma membrane of keratinocytes grown in low calcium, whereas almost no green signal was observed in high calcium (Figs. 3, A and B versus C and D). These results demonstrate first that SR-BI is present in human keratinocytes and second that keratinocyte SR-BI expression decreases in response to calcium-induced differentiation.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   SR-BI protein levels in cultured human keratinocytes decrease with differentiation. Human keratinocytes were grown either in low calcium (0.03 mM) or high calcium (1.2 mM) KGM for 7 days. Equal amounts of protein (50 µg) were applied to each lane, and Western blots were carried out using anti-SR-BI antibody as described under "Experimental Procedures." A representative blot is shown here. The values are the means ± S.E. n = 6 in each group. *, p < 0.001.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Immunofluorescence staining for SR-BI. Human keratinocytes were incubated either in low calcium (0.03 mM) or high calcium (1.2 mM) KGM for 7 days, and then immunofluorescence analysis was carried out as described under "Experimental Procedures." Confocal microscopy visualized a strong green signal for SR-BI localized to the plasma membrane of undifferentiated keratinocytes (A and B). In contrast, almost no green signal was observed in differentiated keratinocytes (C and D). Strong red signals show nuclei revealed by propidium iodide. In C and D the red staining is primarily in the nuclei, but because of the strong intensity of the staining there is some spillover into the cytoplasm.

Cell Association of HDL-CE Is Down-regulated by Calcium-induced Differentiation-- SR-BI mediates the selective uptake of cholesteryl ester from HDL (17). As described above, SR-BI is present in cultured human keratinocytes, and both mRNA and protein levels for this receptor decline with calcium-induced differentiation. To determine whether the calcium-induced decrease in SR-BI alters the cell association of HDL cholesteryl esters by keratinocytes, we next compared [³H]CE-HDL cell association by human keratinocytes grown in either 0.03 or 1.2 mM calcium for 7 days. Keratinocytes were incubated with 50 µg protein/ml of [³H]CE-HDL in the presence or absence of a 25-fold excess of unlabeled HDL for 3 h, and tritium counts in the cell homogenate were measured. Cell association of [³H]CE-HDL significantly declined in high calcium-treated keratinocytes (Fig. 4B), indicating that calcium-induced changes in expression of SR-BI induce parallel changes in cell association of HDL cholesterol.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   A, SDS-PAGE gel of human HDL used in these experiments. B, cell association of radiolabeled HDL in differentiated and undifferentiated keratinocytes. Human keratinocytes were incubated either in low calcium (0.03 mM) or high calcium (1.2 mM) KGM for 7 days and then incubated with 50 µg/ml protein of radiolabeled HDL in the presence or absence of a 25-fold excess of unlabeled HDL for 3 h. The value for specific cell association of HDL was determined by subtracting nonspecific cell association from the total. The values are the means ± S.E. n = 3 in each group. *, p < 0.001. M.W., molecular mass.

SR-BI Is Regulated by Changes in Cellular Cholesterol Levels-- Previous studies in rodent adrenal gland and ovary have shown that decreases in cellular cholesterol levels stimulate SR-BI expression (20, 22). To investigate whether changes in keratinocyte sterol levels also regulate SR-BI expression, we first examined the effects of cellular cholesterol depletion on SR-BI mRNA and protein levels. Human keratinocytes exposed to 10 µM simvastatin, a potent inhibitor of HMG-CoA reductase, displayed a 3-fold increase in SR-BI mRNA levels (Fig. 5) and a 4-fold increase in SR-BI protein levels (Fig. 6), in parallel with more than 90% inhibition of cholesterol synthesis compared with control (control (100 ± 6.7%) versus simvastatin (2.1 ± 0.7%) n = 4, p < 0.001). In contrast, when cellular sterol levels were increased by exposing keratinocytes to 12.5 µM 25-hydroxycholesterol, SR-BI mRNA and protein levels declined significantly (~50%; Figs. 5 and 6). These results indicate that cellular sterol levels regulate SR-BI expression in cultured human keratinocytes.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Sterols regulate SR-BI mRNA levels in cultured human keratinocytes. Human keratinocytes were grown in 0.07 mM calcium KGM to 90% confluence and then treated with either 12.5 µM 25-hydroxycholesterol or 10 µM simvastatin for 24 h. mRNA values were normalized to those of corresponding -actin measured on the same blot and expressed relative to a control value of 1. A representative blot is shown here. The values are the means ± S.E. n = 6 in each group. *, p < 0.05; **, p < 0.001.

View larger version (44K): [in this window] [in a new window]   Fig. 6.   Sterols regulate SR-BI protein levels in cultured human keratinocytes. Human keratinocytes were grown in 0.07 mM calcium KGM to 90% confluence and then treated with either 12.5 µM 25-hydroxycholesterol or 10 µM simvastatin for 48 h. Equal amounts of protein (50 µg) were applied to each lane, and Western blots were carried out using anti-SR-BI antibody as described under "Experimental Procedures." A representative blot is shown here. The values are the means ± S.E. n = 6 in each group. *, p < 0.01; **, p < 0.001.

SR-BI Is Expressed in Murine Epidermis and Regulated by Barrier Requirements-- We next determined whether SR-BI is expressed in epidermis in vivo. As shown in Fig. 7, SR-BI mRNA is present in murine epidermis. Because our laboratory has shown that cholesterol synthesis is required to maintain and restore the epidermal permeability barrier requirements, we next determined whether SR-BI expression is regulated by epidermal barrier requirements. Three hours after acute barrier disruption produced by tape stripping, SR-BI mRNA levels increased 1.5-fold in comparison with untreated controls (p < 0.05) (Fig. 7). To determine whether this increase in SR-BI mRNA levels was due to barrier disruption or other nonspecific effects of tape stripping, we next measured SR-BI mRNA levels following acute barrier disruption by an alternate, unrelated method: repeated acetone swabbing. As observed following tape stripping, barrier disruption with acetone treatment also led to a significant increase in SR-BI mRNA levels at 3 h (p < 0.05) (Fig. 8). Finally, to link these increases in SR-BI mRNA specifically to perturbations in permeability barrier function, we next compared changes in SR-BI mRNA levels after acute barrier disruption followed by immediate, artificial barrier restoration by occlusion with an impermeable membrane. As shown in Figs. 7 and 8, the increases in SR-BI mRNA levels, induced by either tape stripping or acetone treatment, are completely blocked by occlusion, indicating that changes in epidermal SR-BI expression are regulated by permeability barrier requirements.

View larger version (32K): [in this window] [in a new window]   Fig. 7.   Acute barrier disruption by tape stripping increases SR-BI mRNA levels and is reversible by occlusion. 3 h after disrupting the barrier by tape stripping the epidermis was isolated, and Northern blots were prepared as described under "Experimental Procedures." Northern blots were probed for SR-BI mRNA. mRNA values were normalized to those of corresponding cyclophilin measured on the same blot. The values are the means ± S.E. control, n = 16; tape stripping, n = 16; occlusion, n = 7. Control versus tape stripping, **, p < 0.01; tape stripping versus occlusion, *, p < 0.05.

View larger version (40K): [in this window] [in a new window]   Fig. 8.   Acute barrier disruption by acetone treatment increases SR-BI mRNA levels and is reversible by occlusion. 3 h after disrupting the barrier by acetone treatment the epidermis was isolated, and Northern blots were prepared as described under "Experimental Procedures." Northern blots were probed for SR-BI mRNA. mRNA values were normalized to those of corresponding cyclophilin measured on the same blot. The values are the means ± S.E. n = 8 in each group; control versus acetone-treated, *, p < 0.01; acetone-treated versus occlusion, *, p < 0.01.

SR-BI Is Localized to the Basal Layer in Human Epidermis and Up-regulated by Barrier Requirements-- We next determined the localization of SR-BI in human epidermis under basal conditions and following barrier disruption by tape stripping. In normal skin the entire epidermis reveals green staining for SR-BI (Fig. 9A), but the degree of staining is greatest in the basal layer (Fig. 9A, arrows). Three hours after acute barrier disruption produced by tape stripping, the signals for SR-BI were increased (Fig. 9B). These results suggest that SR-BI is predominantly expressed in the basal layer in human epidermis and that barrier disruption increases SR-BI levels.

View larger version (34K): [in this window] [in a new window]   Fig. 9.   SR-BI is expressed in the basal layer of human epidermis and increases following barrier disruption. Immunofluorescence was performed on human skin as described under "Experimental Procedures." A, membrane staining for SR-BI (green signals) was localized especially to the basal layer in control human epidermis (arrows). B, signals for SR-BI were increased in response to permeability barrier requirements. C, negative control. Scale bars, 8 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The cutaneous barrier to water loss is provided by extracellular lipid lamellar membranes in the stratum corneum that contain primarily cholesterol, free fatty acids, and ceramides (1). This lipid lamellar membrane is continually shed, and therefore the viable epidermis has to constantly regenerate this membrane. Moreover, in response to permeability barrier disruption, the viable epidermis initiates a homeostatic repair process, such as the rapid secretion and formation of lamellar bodies and the synthesis of epidermal cholesterol, fatty acid, and ceramide, which provides lipids for lamellar body formation (2, 34), that is responsible for the rapid return of lipid membranes to the extracellular spaces of the stratum corneum leading to the restoration of barrier function. Thus, the epidermis has a large requirement for lipids to continually synthesize these extracellular lamellar membranes. The epidermis is a very active site of lipid synthesis (6, 7). Inhibition of cholesterol, fatty acid, or ceramide synthesis adversely effects barrier homeostasis (3-5), demonstrating that epidermal lipid synthesis is important for providing the lipids required for the formation of the extracellular lipid lamellar membranes. However, inhibition of lipid synthesis only has modest effects on the formation of the extracellular lipid membranes (3, 4, 35), suggesting that extraepidermal sources of lipids also are important. Several other lines of evidence also indicate that extracutaneously derived lipids make a significant contribution to maintaining epidermal barrier function. First, the epidermis requires large quantities of linoleic acid that cannot be synthesized in the epidermis (4, 36, 37) and hence must be obtained from extraepidermal sources. Second, studies have shown that systemically administered labeled cholesterol and fatty acids are delivered to the epidermis (38, 39). Third, the epidermis lacks 5 and 6 desaturase activity (40, 41), and therefore the epidermis presumably must obtain arachidonic acid from other sites. Lastly, plant-derived fatty acids accumulate in the epidermis in certain disease states, such as Refsum's disease (42, 43). Taken together, these observations indicate that extraepidermal sources can contribute to the epidermal lipid pool.

In the present study we demonstrate that SR-BI is expressed in keratinocytes and that the expression is greatest in undifferentiated keratinocytes. The induction of differentiation by incubation in a high calcium medium results in a marked decrease in SR-BI mRNA and protein levels. The functional importance of this change in SR-BI expression is demonstrated by the increased cell association of HDL cholesterol by undifferentiated keratinocytes compared with differentiated keratinocytes. Consistent with our findings in vitro, predominant expression of SR-BI was observed in the basal layer in the epidermis where lipid transport would mainly take place from extraepidermal sources. Taken together, our results presented here suggest that keratinocyte SR-BI plays a role in transport of cholesterol from circulating HDL into the epidermis.

In other tissues, cellular cholesterol levels have been shown to regulate SR-BI expression (20, 22). We also found in this study that keratinocyte SR-BI expression is regulated by changes in cellular cholesterol levels. Keratinocyte SR-BI expression declined in response to increasing cellular sterol levels, whereas inhibiting cholesterol synthesis results in an increase in SR-BI expression. This suggests that SR-BI plays a role in cholesterol homeostasis in keratinocytes. One possible candidate that regulates SR-BI expression in keratinocyte is sterol regulatory element-binding proteins (SRE-BPs). The human SR-BI gene has a number of transcription factor-binding sites including SRE-BPs (21, 44). SRE-BPs are proteolytically cleaved to release the active mature form when intercellular sterol levels decrease and regulate the transcription of key genes for cholesterol homeostasis, such as HMG-CoA reductase, HMG-CoA synthase, and LDL receptor (45-47). Previous studies by our laboratory have shown that SRE-BP 2 is present at high levels, whereas SRE-BP-1 is hardly detected in human keratinocytes and murine epidermis. Both SRE-BP 2 mRNA levels and the proteolytic activation of SRE-BP 2 increase following depletion of intracellular sterols. In contrast, 25-hydroxycholesterol decreases SRE-BP 2 mRNA levels and inhibits the proteolytic conversion to the active form (24). Taken together with our findings in this study, it is likely that SRE-BP 2 is an important regulator of keratinocyte SR-BI expression. In steroidogenic tissues stimulation of cAMP increases SR-BI expression (48, 49); however, in keratinocytes we were unable to demonstrate an effect of cAMP on either SR-BI mRNA or protein levels (data not shown).

Epidermal SR-BI expression increases following barrier disruption by either acetone treatment or tape stripping. That this is an effect of barrier disruption and not a nonspecific effect is shown by the inhibition of the increase in SR-BI by occlusion with a vapor-impermeable membrane that provides an artificial barrier to water movement. Following barrier disruption, there is an increased requirement for lipids in the epidermis that are essential for the formation of new lamellar bodies and the restoration of the permeability barrier (1-5). A marked increase in epidermal cholesterol, fatty acid, and ceramide synthesis in response to barrier disruption is one mechanism by which the keratinocyte is able to obtain the lipids to synthesize new lamellar bodies and repair the permeability barrier function (3-5). One can postulate that the increase in epidermal SR-BI expression following barrier disruption could allow for the increased delivery of cholesterol from extraepidermal sources into epidermis and thereby provide another mechanism by which the keratinocyte obtains the lipids required for the synthesis of new lamellar bodies. Interestingly, immunofluorescence revealed an increase in SR-BI expression over the entire epidermis following barrier disruption. This increase in SR-BI expression could contribute to the rapid formation of new lamellar bodies by delivery of cholesterol directly into highly differentiated keratinocytes where lamellar bodies are synthesized.

In addition to SR-BI, keratinocytes also express LDL receptors (13). The expression of LDL receptors is associated with the ability of cells to internalize LDL (50), and LDL receptor expression is primarily regulated by SRE-BPs (45, 51, 52). The levels of LDL receptors in keratinocytes decrease with differentiation, and LDL receptors have been shown to be present primarily in the basal layer of the epidermis (13-15). Moreover, we have previously shown that LDL receptor mRNA and protein levels are regulated by barrier requirements (10). Thus, both the localization and regulation of LDL receptor and SR-BI expression are very similar in the epidermis. One can postulate that they coordinately mediate the uptake of lipids from circulating lipoproteins. The LDL receptor would allow for the internalization of apolipoprotein B containing lipoprotein particles such as very LDL and LDL, whereas SR-BI would allow for the uptake of cholesterol esters carried primarily in HDL. These two receptors could provide parallel pathways for the delivery of lipids from extraepidermal tissues and the diet to epidermis, thereby facilitating the formation of lamellar bodies and barrier homeostasis.

	ACKNOWLEDGEMENTS

We thank Sally Pennypacker and Debra Crumrine for technical assistance. We are also grateful to POLA Laboratories for support.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants HD 29706, AR 39639, AR 29706, and PO039448 and by Veterans Affairs Research Funding.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Metabolism Section, Dept. of Veterans Affairs Medical Center, 4150 Clement St., Box 111F, San Francisco, CA 94121. Tel.: 415-750-2005; Fax: 415-750-6927; E-mail: kfngld@itsa.ucsf.edu.

Published, JBC Papers in Press, November 13, 2001, DOI 10.1074/jbc.M106445200

	ABBREVIATIONS

The abbreviations used are: HMG-CoA, 3-hydroxy-methylglutaryl coenzyme A; LDL, low density lipoprotein; HDL, high density lipoprotein; SR-BI, scavenger receptor class B type I; CE, cholesterol ester; PBS, phosphate-buffered saline; SRE-BP, sterol regulatory element-binding protein; KGM, keratinocyte growth medium.

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