1,25-Dihydroxyvitamin D3 increases collagen production in dermal fibroblasts

JOURNALOF

Dermatological Science ELSEVIER

Journal of Dermatological Science 8 (1994) 18-24

1,2SDihydroxyvitamin D3 increases collagen production in dermal fibroblasts John Dobakatb, Jacek Grzybowski”, Fu-Tong Liub, Bruce Landona, Marek Dobke*” “Division of Plastic Surgery, University of Calijbrnia San Diego, 200 West Arbor Drive, San Diego. CA 92103-8890. USA bDepartment of Molecular and Experimental Medicine. SBR-4, The Scripps Research Institute. 10666 North Torrey Pines Road, La Jolla, CA 92037, USA

(Received IS November 1993; revision received 14 February 1994; accepted IO March 1994)

Abstract The effect of 1,25_dihydroxyvitamin D3 (1,25-(OH),D,) on proliferation and collagen type I and type III production in cultured human fibroblasts was examined. Previous studies have identified receptors for the vitamin in human dermal fibroblasts, and have suggested the skin to be a target tissue. While many studies examining keratinocyte modulation by 1,25-(OH)2Dj have been undertaken, very few have been performed on dermal fibroblasts. Neonatal foreskin fibroblast cultures were examined for cell number, extracellular collagen accumulation, and collagen mRNA levels, after 5 days exposure to 1,25-(OH)*D, at a concentration of 10e7 M. The vitamin significantly suppressed (P < 0.01) the proliferation of fibroblasts cultured in the presence of serum. Day 5 cell culture supematants showed a significant per cell increase in collagen type I (P < 0.05) and type III (P < 0.01) as measured by ELISA. Type I collagen production in exposed cells was 11.64 + 0.531 &lo6 cell vs. 9.53 + 0.500 &lo6 cells in unexposed cells. Type III collagen production was 0.601 + 0.012 &lo6 cell in exposed cells and 0.247 + 0.008 &lo6 cells in unexposed cells. mRNA levels were increased after a 4-day exposure to 10m7M 1,25-(OH),D, for both type I (2.5-5-fold) and type III (5.5-7.76-fold) collagen. These results suggest a novel effect of increased collagen production by dermal fibroblasts upon exposure to 1,25-(OH)*D3 that is independent of proliferation. Keywordr:

Collagen production;

Dermal Iibroblasts

tive metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2DJ). 1,25-(OH)2D3 has traditionally been

1. Introduction Vitamin

D3 is produced

at the skin, and subse-

quently hydroxylated at the 25 and 1 positions in the liver and kidney, respectively, to form the ac* Corresponding author.

viewed as the major regulator of calcium and phosphate levels through its action on the kidneys and intestines. However, recent evidence has suggested the skin to be a target organ for 1,25-(OH)2D3 [l]. Receptors for 1,25-(OH),D, have been found in

0 1994 Elsevier Science Ireland Ltd. All rights reserved 0923-181 l/94/$07.00 SSDI 0923-1811(94)00307-Z

J. Dobak et al. /J. Dermatol. Sci. 8 (1994) 18-24

the cells of the skin (keratinocytes and fibroblasts), and nuclear localization and DNA binding of receptor bound 1,25-(OH)zD3 has been identified in derma1 fibroblasts [2-41. Normal dermal fibroblast growth is inhibited in a dose-dependent manner (10-6-10-10 M) by 1,25-(OH),D, [5]. 1,25-(OH)2Dj also stimulates the 25-hydroxy D-24R-hydroxylase, an enzyme which metabolizes 25-hydroxyvitamin D, to 24,25-hydroxyvitamin D3 (the significance of the latter compound is unknown) [4]. These effects seen in normal tibroblasts do not occur in fibroblasts from persons with Vitamin D-dependent rickets, a heritable disorder in which there is a missing or defective vitamin D receptor. Hence, the cellular changes observed can be attributed to the 1,25-(OH)zD,. Finally, fibroblasts cultured from skin adjacent to psoriatic lesions show partial resistance to the antiproliferative effects of 1,25(OH),D, seen in normal tibroblasts, suggesting that an abnormal response to 1,25-(OH),D3 may contribute to pathological conditions [6]. The results of previous inquiry into vitamin D effect on dermal fibroblasts suggest a possible regulatory role for the compound and substantiate further inquiries into its effect on these cells. 1,25-(OH),D, has been shown to affect collagen synthesis both positively and negatively in osteoblasts (both cell culture and organ culture) with inhibition of synthesis occurring in the more differentiated form [7-91. Moreover, this regulation is believed to occur at the level of transcription through the action of the receptor complex at upstream activating sequences [IO- 131.Because fibroblasts are also producers of collagen, the present study was undertaken to examine the effect of 1,25-(OH)*D3 on production of type I and type III collagen using ELISA and mRNA analysis. 2. Materials and methods 2.1. Culturing of fibroblasts

Human tibroblasts monolayer cultures were established from foreskins obtained by circumcision. Cells were cultured in 75-cm2 tissue culture flasks (Corning Glass Works, Corning, NY) using growth medium composed of MEM Earle, 10% Fetal Bovine Serum (FBS), and penicillin and streptomycin (all reagents: CORE Cell Culture Facility.

19

University of California, San Diego). The cultures were grown in a humidified incubator containing an atmosphere of 5% CO2 and 95% air. Cultures were harvested using 0.25% trypsin solution (CORE, UCSD). 2.2. Measurement duction

of extracellular

collagen pro-

Fibroblasts from stock cultures were harvested with trypsin, washed with MEM, and suspended in MEM containing 10% FBS, penicillin, streptomycin, and 50 &ml of ascorbate (Sigma, St. Louis, MO) (medium) or medium supplemented with lo-’ M 1,25-(OH)2D3 (kindly provided by Dr. Uskokovic, Hoffman-LaRoche Inc., Nutley, NJ). Cells were then seeded (2 ml) on 6-well tissue culture plates (Corning Glass Works, Corning, NY) in duplicate, at a density of 15 000 cells/cm2. On day 3, cell-culture supernatants from one plate were harvested and kept frozen at -20°C. Cell viability and number were determined by trypan blue exclusion and counting on a hemocytometer. On the duplicate plate, media were changed and vitamin D, dosing was repeated. On day 5 supernatants were harvested and cell viability and number were determined by the same methods. Collagen type I and III content in the supernatants was examined using sandwich enzyme-linked immunosorbent assay (ELISA). 2.3. ELISA ELISA was carried out in 96-well microtiter plates (Fisher Scientific, Fairlawn. NJ). The standard volume added to the wells was 100 ~1. All washes between steps were done with PBS (pH 7.4) containing O.l”%Tween 20 (PBS-T). All incubations, other than color development, were done for 1 h at room temperature. Wells were coated with 100 ~1of affinity purified goat antibodies against collagen type I or III (Southern Biotechnology Associates, Birmingham, AL), diiuted to 10 ,ug/ml in 50 mM carbonate buffer (Sigma) (pH 9.6), and allowed to dry overnight at 37°C. After washing three times with PBS-T, collagen type 1 and type 111standards (Hey], Houston, TX), diluted in PBS-T containing 5 pg/ml of bovine serum albumin (PBS-T-A), were added at concentrations ranging from 0.16 to 5 pgiml. Cell-culture

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J. Dobak et al. /J. Dermatol. Sci. 8 (1994) 18-24

supernatants used for type I determinations were diluted 1:2 for day 3 and 1:4 for day 5 in PBS. Cellculture supernatants for type III were undiluted. Affinity purified biotinylated anti-collagen type I or III antibodies (Southern Biotechnology), diluted in PBS-T-A, were subsequently added in concentrations of 250 ng/ml or 125 @ml, respectively. Wells were then filled with horseradish peroxidase-labeled streptavidin (Southern Biotechnology) diluted in PBS-T-A to a concentration of 200 @ml. For development, 150 jd of a 0.4 mg/ml solution of ophenylenediamine in phosphate citrate buffer (pH 5.0), with 6 ~1 of 30% hydrogen peroxide125 ml of buffer (Sigma) was added. The enzymatic reaction was allowed to proceed for 20 min. Color development was read on a Titertech multiscan spectrophotometer at 450 nm. Statistical differences between the means were assessed using Students ttest analysis. 2.4. mRNA analysis Fibroblasts were grown in 150-cm2 flasks in medium alone or in medium supplemented with 1,25(OH),D3 of 10e7 M (5 flasks for each group). Medium for these experiments did not contain ascorbate. Media was changed on day 3. On day 4, cells were lysed in the flask with a solution of 20 mM Tris (pH 7.4), 10 mM EDTA, 0.1 M NaCl, and 0.5% SDS. Lysate was subject to sheering with a 22-gauge needle and digestion with proteinase K (10 mgml) for 1 h. NaCl was subsequently added to lysate to a final concentration of 0.4 M, and poly(A)+ RNA was isolated by incubation for 2 h with oligo-dT cellulose (Stratagene, La Jolla, CA) (2-3 mg/ml of lysate) suspended in 0.4 M NaCl, 20 mM Tris (pH 7.4), 10 mM EDTA, and 0.2% SDS (loading buffer). The cellulose was hydrated with water and washed twice with loading buffer prior to addition. Following the incubation, the oligo-dT cellulose was washed three times with 1 ml of 0.1 M NaCl 10 mM Tris (pH 7.4), 1 mM EDTA, and 0.2% SDS. mRNA was eluted three times with 1 ml of water, precipitated in ethanol, and centrifuged at 30 000 rev./min for 30 min. Quality of RNA was verified by running non-denatured RNA on a 1% agarose gel in 0.5 x TBE (Tris-borate 0.0045 M, EDTA 0.001 M, pH 8.0). For Northern hybridization, 2 c(g of poly(A)+

RNA was heat-denatured (60°C for 15 min) in 50% formamide and electrophoresed on a 1% agarose gel containing 1 M formaldehyde. RNA was transferred to a nitrocellulose membrane by capillary elution with 20 x SSC (3 M NaCl, 0.3 M NaCitrate). Membranes were vacuum baked at 80°C and hybridized with cDNA probes, generously supplied by Dr. Ramirez (type I pro-l chain, clone Hf667, Mount Sinai Medical Center, New York, NY) and Dr. Crystal (type III pro-l, clone pPB68, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD). A P-actin cDNA probe (clone HHC189, ATCC) was also hybridized in the same manner. Probes were labeled by random oligo-primed Klenow fragment DNA synthesis in the presence of [32P]dATP. Hybridized membranes were washed l-3 times for 20 min in 0.1-0.05 x SSC containing 0.1% SDS at 68°C and exposed to radiographic film. Densitometric analysis was performed on an LKB Ultrascan XL (LKB, Sweden). 3. Results 3.1. 1,25-(OH)2D3

inhibits cell proliferation and enhances collagen production of dermal fibroblasts

Cell counts were performed on days 3 and 5 with a media change on day 3. Cells exposed to 10m7M 1,25-(OH)2D3 showed a significant reduction in cell number (Fig. 1). Data are presented as the average of three repetitions. Because the vitamin was diluted in 100% ethanol, media supplemented with 1,25(OH)2D3 had a final ethanol concentration of 0.1%. This concentration of ethanol was found to have no effect on cell proliferation (data not shown). Cell viability was assessed by trypan blue exclusion. Viability was greater than 98% in control cells and in vitamin-exposed cells. The antiproliferative effects of 1,25-(OH),D, on dermal libroblasts has been previously demonstrated [lo] and a maximal effect was observed on days 3-5. The experiments described here on collagen production were performed on cell-culture days 3-5 because it was assumed that effects related to sterol exposure would occur during this time of maximal growth suppression. Collagen accumulation in supernatants on days 3-5 was quantitated by ELISA. For both type 1 and type III, collagen accumulation corrected for the cell number was significantly

J. Dobak

et al. /J.

Dermatol.

Sci. 8 (1994)

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P
p
0.601

161

a

L

0.247

i

Fig. I. Effect of 10m7M 1.2%(OH),D, on cell number. Cells were seeded at a subconfluent density of I5 000cells/cm*. Cells were counted with a hemocytometer on days 3 and 5. Viability was determined to be greater than 98% in both control cells and vitamin-exposed cells as determined by trypan blue exclusion. Value labels are the average of 3 repetitions, and error bars represent the standard deviation. P values were derived from Students t-test.

p
9.53

11.64 -+-

-+--

Fig. 3. Results of collagen type III accumulation in cell-culture supernatants on days 3-5 as measured by ELBA. Cells were exposed to 1,25-(OH),D, at a concentration of 10e7 M for 5 days with a media change on day 3. Value labels are the average of three repetitions, and are expressed on a per cell basis due to the growth inhibitory effects of the vitamin. Error bars represent the standard deviation. The P value was derived from the Students t-test.

increased on day 5 in the presence of 1,25-(OH),D, (Figs. 2 and 3). On day 3, significant differences were observed in l,25-(OH)2D3-exposed cells for type III (0.948 + 0.63 pgEI06 cell exposed vs. 0.664 + 0.01 rg/106 cell unexposed, P c O.Ol), but no difference was observed for type I (5.65 + 1.49 &lo6 cells exposed vs. 6.08 + 1.95 &lo6 cells unexposed). There was no difference in collagen accumulation between controls and cells exposed to 0.1% ethanol (data not shown). 3.2. 1,2S-(OH),D3 fibroblasts

-

&Ol

Fig. 2. Results of collagen type I accumulation in cell-culture supernatants on days 3-5 as measured by ELBA. Cells were exposedto 1,25-(OH),D, at a concentration of 10m7M for 5 days, with a medium change on day 3. Values are the average of three repetitions, and are expressed on a per cell basis due to the growth inhibitory effects of the vitamin. Error bars represent the standard deviation. The P value was derived from the Students r-test.

upregulates collagen mRNA in

As an initial step in understanding the molecular basis for the observed effect of 1,25-(OH),D, on collagen production, the level of collagen mRNA was analyzed. Ascorbate was eliminated from the medium because of its effect on collagen gene transcription. For both type I and type III, increases in mRNA levels were observed in cells cultured for 4 days in the presence of lo-’ M l,25-(OH)2D3 as compared to controls. No difference was observed in @-actin mRNA which was used as a control. For type I, two bands were detected, 7.2 kb and 5.9 kb in length (Fig. 4). Densitometric measurements

J. Dobak et al. /J.

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Dermaiol. Sci. 8 (1994) 18-24

AUTORADIOGRAPH OF VORTHERN * BLOT

CDCDCD 9.5 kb7.5 kb4.4 kb-

2.4 kb1.4 kb-

Fig. 4. Autoradiography of Northern Blot comparing mRNA between control cells (C) and cells exposed to 1,25-(OH),D, at a concentration of 10m7M (D). Cells were exposed to the vitamin for 4 days with a media change on day 3. Blot was hybridized with cDNA probes corresponding to Type I pro-l(l) [I], type II! pro-l(II1) [2], and &actin [3] mRNA species.

showed a 2.41-fold intensity increase for the 5.9-kb species and a 5.1-fold increase in the 7.2-kb species in cells exposed to 1,25-(OH)2D3. Similarly, two bands, 6.6 kb and 5.8 kb in length, were detected for type III, with a 7.76-fold intensity increase for the 5.8-kb species and a 5.5-fold increase in the 6.6kb species in 1,25-(OH)zD3-exposed cells. The multiple bands detected for the collagen species have been previously described, and represent differences in polyadenylation [15,16]. For j3-actin, a single band, 2.2 kb in length, was detected with essentially no difference in intensity between control cells and 1,25-(OH),Ds-exposed cells as determined by densitometry.

4. Discussion The most important finding of this work is evidence suggesting upregulation of collagen production by 1,25-(OH)2D3 in cultured dermal fibroblasts. Because of 1,25-(OH)zDs’s profound inhibitory effect on cell growth it is important to calculate the collagen production relative to cell number. Correcting for the decreased cell number in the 1,25-(OH)2D3-exposed cells reveals significant increases in collagen production. This data is supported by other data which has shown 1,25(OH),D, to be growth suppressive for a number of cell lines (keratinocytes, promyelocytic leukemia cells, osteosarcoma cells) including tibroblasts [ 16-181. It is unlikely that the reduction in cell number is a toxic effect since there was no difference in cell viability. Additionally, low concentrations of ethanol are not toxic to fibroblasts [19], and there was no difference between controls with ethanol and controls without ethanol. ELISA has been used to quantitate collagen production in cell-culture supernatants and in the study of production of other extracellular matrix components, such as elastin and tibronectin [20-221. It has been shown that in monolayer cultures stabilization of collagen fibers is inefficient, and up to 90% of newly synthesized collagen can accumulate in the culture media [23]. Hence, it is reasonable to examine culture supernatants for quantitation of collagen production. The ELISA assay used here was found to be a sensitive method which allowed type specific identification. The antibodies used were directed against tropocollagen (triple helical molecules with cleaved terminal peptides), and the increased collagen accumulation measured extracellularly is believed to represent enhanced production of intact protein. Further, collagen secreted by the tibroblasts in these experiments was found to be intact by [3H]proline incorporation and SDS PAGE (data not shown). Changes in collagenase activity should not account for the observed differences, since collagenase activity is readily inhibited by serum factors (PIanticollagenase) with a serum concentration of 10% 1241. The data on mRNA analysis strongly suggest that the increased collagen production is at least, in part,

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related to upregulation of collagen mRNA levels. The level of /3-actin mRNA was unaltered by the vitamin. Hence 1,25-(OH),D, appears to have specific enhancement of collagen mRNA expression in cultured dermal tibroblasts. This study does not differentiate between upregulation of transcription and stabilization of mRNA transcripts. Vitamin D has been found to regulate collagen production at the level of transcription in osteoblasts, however many studies demonstrated an inhibitory effect [7,12,13]. Collagen production is also regulated through the stabilization of transcriptional products [25]. No difference in mRNA expression was observed at 24 h (data not shown), which suggests the hormone may act through transcript stabilization during the 4-day culture period. However, given the data for 1,25-(OH),D, regulation of the collagen promoter in osteoblasts, and the fact that 1,25-(OH)zD, dosing was repeated just prior to the mRNA analysis, some transcriptional regulation seems likely. Finally, control of newly synthesized collagen accumulation extracellularly occurs through alterations in intracellular degradation [26]. CAMP appears to mediate this degradation, with increased levels resulting in increased destruction [271. There is some evidence to suggest that 1,25(OH)2D3 potentiates decreases in CAMP [281. Hence 1,25-(OH)*D3 may also be stabilizing collagen intracellularly, resulting in increased accumulation extracellularly. It is interesting to speculate that 1,25-(OH)2D3 may modulate collagen production in the dermis. For example, in a healing wound, where libroblasts are stimulated by a multitude of growth factors (similar to the conditions of serum cultured cells), the vitamin may enhance collagen production and improve tensile strength, while at the same time keep fibroblast growth under control. Activated macrophages are known to hydroxylate 25-hydroxyvitamin D3 at the 1 position to form the active metabolite, and their presence in the healing wound may supply fibroblasts with the active vitamin form 1291. With the rapidly developing knowledge on molecular mechanisms of healing processes (‘normal’ and pathological), libroblast and collagen role in control of neoplastic cells invasion, need for studying of factors with modulating potential, such as vitamin Ds, has to be appreciated [30,31].

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5. Acknowledgement The authors would like to thank Dr. Uskokovic of Hoffman-LaRoche for his generous supply of 1,25-(OH)2D3, and Dr. Crystal and Dr. Ramirez for the collagen probes. This work was supported by the Plastic Surgery Research Foundation of San Diego, UCSD Academic Senate, and National Institutes of Health grant 5T32 AR07144, publication number 7700-MEM Scripps Reserarch Foundation. 6. References I

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