Vitamin D stimulates DNA synthesis in alveolar type-II cells

ELSEVIER

Biochimica et BiophysicaActa 1221 (1994) 159-166

Biochi~ic~a ct BiophysicaA~ta

Vitamin D stimulates D N A synthesis in alveolar type-II cells Jeffrey D. Edelson a,., Shirley Chan a, Davinder Jassal b Martin Post b A. Keith Tanswell b a Department of Medicine, St. Michael's Hospital, Rm. 2120B, 30 Bond Street, Toronto, Ontario, Canada M5B 1W8 b Department of Paediatrics, the Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada M5G 1X8

(Received 8 September 1993)

Abstract

Alveolar type-II cells are responsible for alveolar epithelial cell proliferation during growth and development and in response to lung injury. Based on the observation of abnormal lung development in rachitic rat pups and the expression of receptors for vitamin D by fetal alveolar epithelial cells, the present study examined the influence of 1,25-dihydroxy vitamin D (DHD) on the proliferation of primary cultures of fetal, neonatal and adult alveolar epithelial cells. The ontogony of vitamin D responsiveness was examined, using fetal (days 18, 19 and 22 = term), neonatal (days 7 and 18) alveolar epithelial cells as well as adult alveolar type-II cells. Maximal stimulation of [3H]thymidine incorporation occurred in neonatal d18 cells: (250 + 4.8%, n = 4, P < 0.05). Incubation of adult type-II cells, in the presence of 10 - 9 M DHD increased thymidine incorporation into DNA (149.1 + 33.2%, mean + S.E., n = 3, P < 0.001) compared to control cells maintained in basal medium. Exposure to DHD also increased thymidine incorporation after stimulation with a mixture of conventional progression factors (insulin (10 /~g/ml) (I), cholera toxin (10/~g/ml) (C) and EGF (20 ng/ml) (E)) (349.4 + 42.9% vs. 213.5 + 23.6%, n = 6, P < 0.005). Autoradiographic labeling indices of adult type-II cells increased from 3.1 5: 0.6% for cells cultured in basal medium to 7.2 + 1.7% in cells exposed to DHD from the time of plating and I, C, E from 20-68 h in culture (n = 4, P < 0.05). Although no increase in the number of adult type-II cells was observed in these experiments, flow cytometric analysis of nuclear DNA content revealed an increased proportion of cells in the S and G2 phases of the cell cycle (basal: S = 2.6%, G 2 / M = 3.0%, DHD + GF: S = 4.7%, G 2 / M = 5.6%, P < 0.05 for each comparison). These data demonstrate that vitamin D3 is a growth factor for alveolar type-II cells and suggest the possibility that local elaboration of vitamin D may provide a novel mechanism of modulation of epithelial proliferation in the context of lung development and repair. Key words: Vitamin D; Type II cell; Proliferation; PDGF; Alveolar epithelium

1. Introduction

In addition to its well recognized role in bone and mineral metabolism, the secosteroid 1,25-dihydroxy vitamin D ( D H D ) modulates a variety of other cellular activities [1]. The possibility that D H D is important in lung development is suggested by the observation that rachitic rat pups, born to vitamin D deficient mothers, demonstrate normal lung weights but decreased lung compliance, consistent with abnormal alveolar development [2]. Fetal alveolar epithelial cells express receptors for D H D [3], and fetal lung explants decrease glycogen content and increase surfactant synthesis and secretion after exposure to 10 -9 M D H D [4,5]. These

* Corresponding author. Fax: + 1 (416) 864-5870. 0167-4889/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0167-4889(93)E0193-4

data suggest that D H D may modulate alveolar epithelial function in the developing lung. Proliferation of the alveolar epithelium is important in maintaining the integrity of the lung epithelium during growth, development and in response to lung injury [6-10]. Proliferation of alveolar epithelial cells in vitro is modulated by the culture substratum [11,12], soluble factors present in the culture system [13-15] and macrophage-derived growth factors [16,17]. Recognized macrophage-derived growth factors include platelet-derived growth factor, transforming growth factor beta, basic fibroblast growth factor and transforming growth factor alpha [18]. Alveolar macrophages are capable of activating 25-hydroxyvitamin D to the metabolically active 1,25-dihydroxyvitamin D3 form ( D H D ) [19,20], suggesting the possibility that the elaboration of D H D by activated macrophages may provide

16(/

J.D. Edelson et al. / Biochirnica et Biophysica Acta 1221 (1994) 159-166

an additional paracrine influence on alveolar epithelial function. In vitro data demonstrate that DHD can both stimulate [21,22] or inhibit [23,24] cell proliferation. These effects vary with the cell type under examination and may also be concentration dependent [25]. The present study examined the influence of DHD on alveolar epithelial proliferation in fetal and neonatal alveolar epithelial cells and adult rat alveolar type-II cells.

2. Materials and methods

Reagents Porcine pancreatic elastase, type I DNase (2367 U / m g ) and CLS 1 collagenase (131 U / m g ) w e r e purchased from Worthington Biochemical (Freehold, N J). Gentamycin, insulin, epidermal growth factor (EGF), bovine serum albumin, ergocalciferol and prostaglandin E~ were obtained from Sigma (St. Louis, MO) and DHD was purchased from Biomol Research Lab. (Plymouth Meeting, PA). [3H]Thymidine was purchased from Amersham, Oakville, ON. Powdered culture media, fetal bovine serum, Hepes buffer, trypsinEDTA and antibiotics were obtained from Gibco-BRL Life Technologies (Burlington, ON). For those studies in which cells were maintained in defined serum-free medium, we obtained insulin, selenous acid, transferrin and laminin from Collaborative Research (Bedford, MA). Poly(D-lysine) was obtained from BoehringerMannheim (Mannheim, Germany). Cholera toxin was from Calbiochem (La Jolla, CA). Porcine platelet-derived growth factor (PDGF) was from R + D Systems (Minneapolis, MN).

Isolation of fetal distal lung epithelial cells Primary cultures of distal lung epithelial cells were prepared from specific pathogen-free Wistar rats as previously described [26]. Briefly, mixed cell suspensions were obtained from fetal rat lung homogenates by sequential incubation with 0.1% (w/v) of trypsin, 0.001% DNase and 0.1% coUagenase. Fibroblasts were removed by differential adherence and sedimentation. Epithelial cells were then plated at 3.104 cells/cm 2 for 24 h to allow attachment to culture substrata, in the presence of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum. After 24 h, the serum containing media was removed and cells maintained in DMEM containing insulin (50 t~g/ml), bovine serum albumin (50/~g/ml), selenous acid (4 ng/ml), reduced glutathione (500 n g / ml), soybean trypsin inhibitor (100/~g/ml), transferrin (5 /xg/ml), Hepes buffer (2.6 m g / m l ) and cholera toxin (5/zg/ml). This mixture was based on previously described serum-free media for fetal distal lung epithelial cells [26] and adult type-II pneumocytes [15] and

was used for both cell types in studies of the ontogeny of the cellular response to DHD. Cells were maintained either on a poly(D-lysine) or a laminin substratum applied according to the manufacturer's instructions. Laminin was applied in distilled H20 at 2 /zg/ cm 2 and air dried. Poly(D-lysine) was applied at 2.5 /zg/cm 2 in distilled water and allowed to stand at room temperature for 5 min. Residual solution was removed and substrata washed twice with distilled water. Prior to use, both substrata were incubated in medium with 1% BSA overnight to block nonspecific binding sites.

Isolation and culture of neonatal and adult alveolar type-H cells Type-II cells were isolated from normal male adult Sprague-Dawley rats and from neonatal rats of 7 and 18 days of age using the method of Dobbs [27]. Type-II cells were separated from the alveolar basement membrane by incubation with porcine pancreatic elastase followed by removal of macrophages by differential adherence on Fc-coated Petri dishes (Fisher Scientific, Ottawa, ON). Type-II cells were identified on cytospun samples and in culture dishes by histochemical staining for alkaline phosphatase [28]. Cells were plated in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine (Gibco), 10 /zg/ml gentamycin, 100 U / m l penicillin G, 100 /zg / m l streptomycin, 0.25 ~ g / m l amphotericin B (D10) at an initial seeding density of 5.105 cells/cm 2 in 24-well culture dishes (Falcon Multiwell, Becton Dickinson Labware, Lincoln Park, N J).

Thymidine incorporation Thymidine incorporation into DNA by type-II cells was studied as previously described [13,15]. Type-II cells were plated onto tissue culture plastic in D10 with or without media supplements. 20 h after plating, the media was aspirated and replaced by D10 containing 1 /zCi/ml of [3H]thymidine (specific activity 5 Ci/mmol), in the presence or absence of media supplements. 68 h after plating, media were aspirated and discarded. The cells were washed and DNA was precipitated. For studies involving fetal and neonatal cells, isotope was added 48 h after plating and the duration of the incubation was 24 h.

Specificity of DHD type-H cell binding Type-II cells were plated at an initial seeding density of 5 • 105 ceUs/cm 2 in 24-well culture dishes and placed in a 37°C incubator overnight. The cells were washed three times with 1% albumin in PBS. The monolayers were treated with 1 nM [aH]DHD (NEN Dupont, Markham, Ont.) in the presence or absence of 1000-fold excess cold DHD (Biomol research, Plymouth Meeting PA). The cells were incubated for 5 h

J.D. Edelson et al. / Biochimica et Biophysica Acta 1221 (1994) 159-166

at 4°C in a covered box and washed five times in 1% albumin-PBS. 500 ~1 of lysis buffer (1% Triton X-100, 10 mM glycine, 20 mM Hepes, pH 7.4) was added to each well and incubated overnight at 4°C. The suspension was then transferred to a scintillation vial and counted in the beta counter.

Cell number Cells were removed from culture substrata with 0.05% trypsin/0.5 mM EDTA. Trypsin was inactivated by the addition of 10% fetal bovine serum and cell aggregates dispersed by passing the cell suspension through a 25 gauge needle. Cells were stained with 0.4% crystal violet and counted with a Neubauer hemocytometer (Superior, Germany). Autoradiography Autoradiographs were prepared by staining cells with tannic acid [29] prior to application of autoradiographic emulsion as previously described [13]. Type-II cells were plated onto 35-mm tissue culture dishes in either D10 or D10 supplemented with DHD at an initial plating density of 2.5" 105 cells/cm z. 20 h after plating, media were aspirated and replaced with D10 that contained 1 /zCi/ml [3H]thymidine in the presence or absence of I, C, E, DHD. 68 h after plating, cells were washed and fixed with 1.5% glutaraldehyde (w/v in PBS, 4°C, 15 min) and then with 1% osmium tetroxide (4°C, 90 min) and stained with 1% tannic acid (4°C, overnight). Cells were then washed with PBS, distilled water and coated with a 1 : 1 dilution of NTB II emulsion (Terochem, Eastman Kodak, Rochester, NY). Culture dishes were stored in a light-protected box for 10 days at 4°C. Autoradiographs were developed and the labeling index determined by counting the number of type-II cells that had incorporated [3H]thymidine. (Cells that contained four or more tannic-acid-labeled inclusion bodies were scored as type-II cells.) 500 type-II cells per dish were counted. Flow cytometry DNA content was measured in suspensions of nuclei prepared from cultured type-II cells using the method of Vindelov et al. [30]. Cultured type-II cells were scrapped into buffer containing 40 mM citrate, 5% DMSO and frozen in dry-ice-chilled ethanol [31]. Nuclei were then thawed and stained with propidium iodide and analyzed using an Epics V flow cytometer (Coulter, Hialea, FL). The proportion of cells in G0/G1, S and G 2 / M stages of the cell cycle were determined by electronic integration of the areas under the appropriate DNA histograms. EGF binding analysis Specific binding of [125I]EGF was determined in type-II cells by plating 1 • 106 type-II cells in 24-well

161

tissue culture plastic wells in D10 or D10 supplemented with DHD. 20 h after plating, the wells were washed with 1% albumin in PBS. Type-II cells were incubated with [125I]EGF in the presence or absence of a 1000-fold excess of unlabelled EGF for 5 h, 4°C. The ceils were washed five times with 1% albumin in phosphate-buffered saline and 1 ml of lysis buffer (1% Triton X-100, 10 mM glycerol, 20 mM Hepes, pH = 7.4) was added and cells lysed overnight. Radioactivity of cell lysates was analyzed using a scintillation counter.

Statistics All assays were performed on paired samples from each experimental assay condition, using cells isolated from n > 3 cell isolations. Results are expressed as the mean _+standard error of the mean. Statistical significance (P < 0.05) was determined by an analysis of variance, followed by assessment of differences using Dunnet's two-sided test [32] or Duncan's multiple-range test [33].

3. Results

Initial studies examined the ontogeny of type-II cell responsiveness to vitamin D. A defined culture system, in which alveolar epithelial proliferation has been demonstrated was utilized [26]. Distal fetal lung epithelial cells were isolated from rat lungs at 18, 19 and 22 days gestation (F18, F19, F22), which represent the pseudoglandular, canalicular and saccular stages of fetal lung maturation respectively; and at 7 (PN 7) and 18 days (PN 18) after birth and from adult animals. Cells were exposed to 1 nM DHD for 24 h under defined, serum free conditions on poly(D-lysine) substrata (Fig. 1). Maximal stimulation of [3H]thymidine incorporation occurred in PN 18 cells: (250 _+48%, n = 4, P < 0.05). There was no effect on early neonatal (PN 7) cells and small effects on fetal cells, which did not achieve statistical significance. Although the interactions of alveolar epithelial cells with extracellular culture matrix can influence the cellular response to growth factors, [11,12], the responses of alveolar type-II cells to vitamin D was similar when studied in cells cultured either on laminin or poly(Dlysine) substrata. Similar to cells cultured on poly(Dlysine) substrata, PN 18 cells showed maximal responses (335 + 67%, n = 4, P < 0.05) when maintained on laminin (Fig. 2). The ability of DHD to augment DNA synthesis by adult alveolar type-II cells was examined (Fig. 3). The purity of the primary cell populations of adult type-II cells on day 1 (20 h after plating) for cells used in these experiments ranged from 87 to 97% (92 + 2.9%, mean _+S.E.), as assessed by alkaline phosphatase staining. Growth factors, such as insulin (I, 10/zg/ml), cholera

J.D. Edelson et al./Biochimica et Biophysica Acta 1221 (1994) 159-166

162

toxin (C, 10 ~ g / m l ) and E G F (E, 20 n g / m l ) increased thymidine uptake by 213.5 + 23.6% c p m / m g protein (mean + S.E., n = 12) compared to control cells maintained in D10 (n = 12, P < 0.001). Exposure to D H D (10 -9 M) increased both basal (149.1 + 33.2%, n = 3, P < 0.001) and I, C, E stimulated (349.4 +__42.9%, n = 6, P < 0.005) thymidine uptake. Dose-response studies for D H D (Fig. 4) demonstrated that there is a dose-response relationship for stimulation of [3H]thymidine incorporation into D N A by adult alveolar type-II cells and that 10 -9 M is the optimal concentration observed. In contrast, vitamin D2, (ergocalciferol) did not increase [3H]thymidine incorporation. The ability of D H D to bind specifically to cultured alveolar type-II cells was examined. 3Hlabelled D H D binding was reduced in the presence of 1000-fold excess of cold DHD, suggesting specific binding of D H D accounted for 28.4 + 4.8% of the cell-associated [3H]DHD (mean + S.E., n = 4). To confirm that the observed increase in thymidine incorporation was due to D N A synthesis by adult alveolar type-II cells, autoradiographic studies and thymidine incorporation experiments were performed under similar conditions. [3H]thymidine incorporation corre-

....

300

i

I

0 n,. iz 0 o z

**--

200

< 0a . 0 0 z .1

t

400 A .I

i I

0 I--

z O O

30O

z

_o 200 0 0z Ill

z

i >-r

100

D18

D19 FETAL

D22

07

D18

ADULT

POSTNATAL

Fig. 2. Incorporation of [3H]thymidine into alveolar type-II cells. Cells were exposed to 10 - 9 M D H D for 24 h u n d e r defined, serum-free conditions on a laminin culture substrate and exposed to isotope from 48 to 72 h after plating. Vertical bars represent S.E. of the m e a n s of three separate experiments. * Indicates that day 18 postnatal cells are significantly different from all other groups; • * adult cells are significantly different from day 18 fetal and day 18 fetal, day 7 and day 18 postnatal cells ( P < 0.05).

J

I,,"

z

I!

100

_0 X ',r I--

D18

D19 FETAL

022

D7

D18

ADULT

POSTNATAL

Fig. 1. Incorporation o f [ 3 H ] t h y m i d i n e into rat alveolar epithelial

cells. Cells were exposed to l0 - 9 M D H D for 24 h u n d e r defined, serum-free conditions on a poly(D-lysine) culture substrate, and exposed to isotope from 48 to 72 h after plating. Vertical bars represent S.E. of the m e a n of > 5 separate experiments. * Indicates that day 18 cells are significantly different from D18, D19, D22 fetal and D7 postnatal cells; * * denotes a significant difference between adult and day 7 cells ( P < 0.05).

lated with an increase in labeling index from 3.1 + 0.63%, in control cells to 7.2 + 1.7%, (n = 4, P < 0.05) in cells exposed to D H D from the time of plating and then stimulated by I, C, E (Fig. 5). T h e r e was no increase in cell number observed in adult type-II cells under these conditions (data not shown). Exposure of day 22 fetal epithelial cells to 10 -9 M D H D for a period of 5 days caused an increase in cell number from 3362 + 657 cells/well (in medium containing insulin) to 7845 + 2318 (in medium containing insulin and DHD, mean + S.E., n = 4, P < 0.05). To evaluate the effects of D H D exposure on the cell cycle position of type-II cell populations, D N A content was evaluated in nuclei isolated from adult type-II cells under isopyknic conditions. After 68 h in culture on plastic substrata in the presence of D10, 94.5 + 0.6% (mean + S.E., n = 5) of cells were identified in the G 0 / G 1 phase of the cell cycle, 2.6 + 0.4% were in the S phase and 3.0 + 0.2% were in the G 2 / M phase (Table 1). Incubation of type-II cells with 10 -9 M D H D followed by the addition of growth factors at 20 h significantly increased the proportion of cells in both S and G 2 / M phases of the cell cycle (S = 4.7 + 1.5%, P < 0.05, G 2 / M = 5.6 + 0.9%, P < 0.01).

J.D. Edelson et al. / Biochimica et Biophysica Acta 1221 (1994) 159-166

C+D3+I,C,E

C+I,C,E

C+D3 I i

C

m 0 100 200 300 400 THYMIDIHE IHCORPORATION (% CONTROL)

Fig. 3. Incorporation of [3H]thymidine into TCA-insoluble DNA of alveolar type-II ceils. 20 h after plating, cells were labeled with 1 mCi/ml of [3H]thymidinefor a period of 48 h. C: cells maintained in D10. D3 denotes cells maintained in 1,25-dihydroxyvitamin D (10-9M); I: cells exposed to insulin (10/~g/ml); C: cholera toxin (10 /zg/ml) and E: EGF (20 ng/ml) from 20 to 68 h after plating. Horizontal bars denote S.E. of the means of 12, 6, 12, 6 separate experiments, respectively. Each group is statistically distinct from each other group at a P < 0.05 level.

163

siveness to D H D was observed in cells at postnatal day 18. This stimulation is at least additive with c o n v e n tional progression type growth factors (I, EGF). The stimulatory effect of D H D is not dependent on culture substratum and is suppressed by the presence of PDGF-BB in adult cells. These results are of potential importance with regard to epithelial proliferation in growth, development and repair. D H D induced epithelial proliferation may be regulated by means of D H D supply, changes in vitamin D induced signal transduction or suppression of D H D effects by PDGF-BB. The supply of D H D is largely regulated by renal hydroxylation at the 1 position of relatively inactive hepatic-derived 25-OH vitamin D. It is possible that pulmonary hypoplasia accompanying renal agenesis, or uremia might be due in part to abnormal regulation of epithelial proliferation, similar to the evident epithelial hypoplasia that occurs in rat pups rendered rachitic [35]. In fetal life the placenta may also be an important source of D H D [36]. In the postnatal lung, alveolar macrophages, which are capable of 1-hydroxylating the 25-OH D [19,20], represent a potential source of DHD. A second possible level for modulation of vitamin D induced epithelial cell proliferation is at the level of receptor density. Although the present commmunica-

200 190 180

170 0

Incubation with D H D did not increase specific binding of E G F to adult alveolar type-II cells. In control cells 81.3 _+ 7.3% (mean + S.E., n = 5) of [lzSI]EGF was specifically bound, whereas in cells exposed to DHD, 77.9 + 6.4% of added E G F was bound specifically ( n - - 5 , P = n.s.). Based on the observation that PDGF-BB inhibits the growth stimulating effects of vitamin D H D in smooth-muscle cells [34], type-II cells were exposed to 20 n g / m l of PDGF-BB. Exposure to PDGF-BB inhibited the response to D H D in adult type-II cells (Fig. 6), but not in fetal cells (data not shown).

160

re" z.- 15o Z O ¢.) 1 4 0 a~

130

~.

120

O

F-

110 10o

oel

90

0

60

n,-

u ~,"

70

u.i

60

9

so

I

4O

I,,,-

30

Z

20

4. Discussion The present study demonstrates that D H D acts to increase thymidine incorporation into D N A in late neonatal and adult alveolar type-II ceils. The increase in thyrnidine incorporation is accompanied by an increase in type-II cell labeling index and progression into active stages of the cell cycle. T h e developmental profile of vitamin response shows that maximal respon-

]

-

10

10-12

10"11 OHO

10-10

10-9

CONCEHTRAT|OH

10 .8

10-7

(M)

Fig. 4. Dose-response relationship for ],25-dibydroxy vitamin D (10-9M) (DHD). Primary cultures of adult type-II cells were incubated with the indicated concentration of DHD and exposed to [3Hlthymidine between 20 and 72 h in culture as described in Section 2.

164

J.D. Edelson et al. / Biochimica et Biophysica Acta 1221 (1994) 159-160 10

400

9

-!

on,8

-

I

iz

o

I

O

300

-1 !

Z

6

o

-

I,< nO a,

5

~

4

0 0 Z

,-

3

w Z a

>.

~"

-

i

2

200 --!

100

-

J

1

i

0

o

- -

C

D3 + I,C,E

Fig. 5. Labeling index of cultured alveolar type-II cells under control (C) or after exposure to DHD and growth factors (see Fig. 3 for abbreviations). 20 h after plating, cells were exposed to 1 mCi/ml of [3H]thymidine for 48 h. Cells were stained with tannic acid/ polychrome and autoradiographed. Vertical bars represent S.E. of the means of four separate experiments.

tion demonstrates specific binding of D H D to adult rat type-II epithelial cells, there has as yet been no characterization of a hormonally regulated receptor for D H D on adult alveolar type-II cells [3]. A third level whereby the effects of vitamin D may be modulated is suggested by the in vitro inhibition of D H D induced thymidine incorporation by P D G F - B B , as observed in smoothmuscle cells [34]. The ontogeny of P D G F - B m R N A [37] shows an increase in P D G F expression in the 7 days after birth and a subsequent decline to adult levels by 14 days after parturition. However, in the

Table 1 Cell cycle analysis Conditions

n

G0/G1

S

G2/M

C GF DHD+GF

5 5 5

94.5±0.6 90.7±3.7 89.8±2.4*

2.6±0.4 5.7±2.9 4.7±1.5"

3.0±0.2 4.3±0.8 5.6±0.9**

Effects of vitamin D and growth factors on cell cycle position of adult rat alveolar type-II cells. Adult rat alveolar type-II cells were maintained in basal medium (C) or in the presence of 10-gM DHD (1,25 dihydroxy vitamin D) cells were exposed to potential growth factors (insulin 10 ixg/ml, cholera toxin 10/~g/ml, EGF 20 ng/ml.) for 20 to 68 h, at which time cells were lysed and nuclear DNA stained with propidium iodide and analyzed by flow cytometry. All values are expressed a mean + S.E. * P < 0.05. ** P < 0.01.

i

C

I

I/D3 I/D3/PDGF

Fig. 6. Incorporation of [3H]thymidine into adult alveolar type-lI cells. Cells were exposed to I (insulin, 10 izg/ml), D3 (DHD, 10-9M) or I, D3 and PDGF (PDGF-BB 20 ng/ml) under defined, serum-free conditions in a poly(D-lysine)culture substrate. Vertical bars represent the S.E. of the means of three separate experiments. * Significantly different from control conditions; * * significantly different from I.

absence of added PDGF-BB, the present in vitro studies demonstrate that alveolar epithelial responsiveness to D H D peaks at postnatal day 18, by which time in vivo type-II cell division is essentially complete [38]. D H D and P D G F - B B may represent opposing influences in a system of developmentally coordinated modulation of alveolar epithelial proliferation in the developing lung. Previous studies have documented a disparity between thymidine incorporation and change in cell number in vitro cultures of adult type-II cells [39-41]. Clement et al. documented increased thymidine incorporation without an increase in either cell number or cellular D N A content and proposed that reduced stability of D N A account for these findings [42]. Alternatively, Uhal and Rannels, using flow cytometric techniques to study cultured rat type-II cells, observed an increase in the proportion of G 2 / M phase cells 48 h after plating, but no increase in cell number, consistent with a late cell cycle block [43]. The demonstration of an increase in the proportion of binucleated type-II cells [44] is also consistent with a late phase cell cycle block. The present study, demonstrates a 3.5-fold increase in thymidine incorporation (comparing control to maximum stimulatory conditions), which was associated with a doubling of autoradiographic labeling index

J.D. Edelson et al. / Biochimica et Biophysica Acta 1221 (1994) 159-166

and an approximate doubling of cell populations in the S and G 2 / M phases of the cell cycle of cultured adult type-II cells. The failure to observe an increase in cell number under these conditions is consistent with a cell cycle block occurring after the G 2 / S stage of the cell cycle or an increase in proliferation limited to a subpopulation of type-II cells. We therefore extended these observation to a defined culture system in which adult [15] and fetal alveolar epithelial cells undergo cell proliferation [26]. In this serum-free defined system, adult and late neonatal but not fetal or early neonatal alveolar epithelial cells demonstrate both an increase in thymidine incorporation after stimulation with D H D and a significant increase in cell number. There are several possible mechanisms by which D H D may modulate cell proliferation. In a bone-derived cell line, D H D increases expression of EGF receptors [45]. However, incubation with D H D did not increase specific EGF-receptor binding in the present study. Taniguchi has demonstrated that DHD enhances the synthesis of transferrin, a potent lung epithelial mitogen [46]. However, the presence of 5/~g/ml of transferrin in the present culture system renders induction of transferrin synthesis an unlikely explanation for the increase in thymidine incorporation noted in the present study. Some of the biologic activities of DHD may be explained by the activation of protein kinase C [47]. Recent studies have demonstrated stimulation of insulin-like growth-factorbinding proteins by DHD [48,49]. In view of the importance of insulin-like growth factors (IGF) in alveolar epithelial proliferation and the ability of IGF-binding protein 3 to enhance cellular responses to IGF, these data suggest a mechanism by which D H D may augment proliferation of alveolar epithelial cells and explain the additive effects of insulin and DHD in the present culture system. Macrophages are important modulators of alveolar epithelial cell proliferation in the post-natal lung. Shami related influx of inflammatory cells (primarily macrophages) to subsequent alveolar epithelial cell proliferation in response to injury [50]. The ability of alveolar macrophages to hydroxylate 25-hydroxyvitamin D to the metabolically active 1,25-dihydroxyvitamin D3 form (DHD) [51,52], suggests the possibility of a local alveolar source of this growth factor in post-natal or injured animals. Previously recognized macrophageproduced growth factors include platelet-derived growth factor, transforming growth factor beta, basic fibroblast growth factor and transforming growth factor alpha [18]. Leslie and colleagues observed that exposure to macrophage conditioned media or co-culture with macrophages augmented incorporation of [3H]thymidine by adult rat type-II cells [16]. Brandes and Finkelstein demonstrated that stimulated (but not quiescent) macrophages produced a factor that enhanced

165

[3H]thymidine uptake by adult rabbit type-II cells [17]. The factor was heat labile, resistant to reducing and acid conditions, with a molecular weight above 25 kDa. Alveolar macrophages also stimulate proliferation of bovine bronchial epithelial cells [53]. Since alveolar macrophages can activate 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D the elaboration of DHD might provide an additional paracrine mechanism by which alveolar macrophages modulate alveolar epithelial proliferation. These data demonstrate that vitamin D3 is a growth factor for type-II epithelial cells. They suggest the possibility that the elaboration of vitamin D, may represent a novel mechanism for the regulation of epithelial cell proliferation either in the context of postnatal lung growth and development or the repair of the epithelium in response to injury.

5. Acknowledgements

The authors thank Jonathan Plumb for excellent technical assistance. This work was supported by the Ontario Thoracic Society and the Medical Research Council of Canada (grants: MT-10545 (JDE), MT-7867 (AKT)).

6. References [1] Reichel, H., Koeffler, H.P. and Norman, A.W. (1989) N. Engl. J. Med. 320, 980-990. [2] Gaultier, C., Haft, A., Balmain, N., Cuisinier-Gleizes, P. and Mathieu, H. (1984) Am. Rev. Respir. Dis. 130, 1108-1110. [3] Nguyen, T.M., Guillozo, H., Marin, L, Cufour, M.E., Tordet C., Pike, J.W. and Garabedian, M. (1990) Endocrinology 127, 17551762. [4] Marin, L., Dufour, M.E., Tordet, C. and Nguyen, M. (1990) Biol. Neonate 57, 257-260. [5] Marin, L., Dufour, M.E., Nguyen, T.M., Tordet, C. and Garabedian (1993) Am. J. Physiol. 265, L45-L52. [6] Witschi, H. (1976) Toxicology 5, 267-277. [7] Kapanci, Y., Weibel, E.R., Kaplan, H.P. and Robinson, F.R. (1962) Lab. Invest. 20, 101-118. [8] Adamson, I.Y.R. and Bowden, D.H. (1974) Lab. Invest. 30, 35-42. [9] Evans, M.M., Cabral, L.J., Stephens, R.J. and Freeman, G. (1975) Exp. Mol. Pathol. 22, 142-150 [10] Holm, B.A., Matalon, S., Finkelstein, J.N. and Notter, R.N. (1988) J. Appl. Physiol. 65, 2672-2678. [11] Rannels, S.R., Fisher, C.S., Heuser, L.J. and Rannels, D.E. (1989) Am. J. Physiol. 253, C759-C765. [12] Rannels, S.R., Yarnell, J.A., Fisher, C.S., Fabisiak, J.P. and Rannels, D.E. (1987) Am. J. Physiol. 253, C835-C845. [13] Leslie, C.C., McCormick-Shannon, K., Robinson, P.G. and Mason, R.J. (1985) Exp. Lung Res. 8, 53-66. [14] Leslie, C.C., McCormick-Shannon, K. and Mason, R.J. (1990) Am. J. Respir. Cell Mol. Biol. 2, 99-106. [15] Tanswell, A.K., Byrne, P.J., Han, R.N.N., Edelson, J.D. and Han, V.K.M. (1991) Am. J. Physiol. 260, L395-LA02.

166

J.D. Edelson et al. /Biochimica et Biophysica Acta 1221 (1994) 159-166

[16] Leslie, C.C., McCormick-Shannon, K., Cook, J.L. and Mason, R.J. (1985) Am. Rev. Respir. Dis. 132, 1246-1252. [17] Brandes, M.E. and Finkelstein, J.N. (1989) Am. J. Respir. Cell Biol. 2, 233-234. [18] Pierce, G.F. (1990) Am. J. Respir. Cell Mol. Biol. 2, 233-234. [19] Barbour, G.L., Coburn, J.W., Slatopolsky, E., Norman, A.W. and Horst, R.L. (1981) N. Engl. J. Med. 305, 440-443. [20] Reichel, H., Koeffler, H.P., Barbers, R. and Norman, A.W. (1987) J. Clin. Endocrinol. Metab. 65, 1201-1209. [21] Mitsuhashi, T., Morris, R.C., Jr. and Ives, H.E. (1991) J. Clin. Invest. 87, 1889-1895. [22] Freake, H.C., Marcocci, C., Iwasaki, J. and Maclntyre, I. (1981) Biochem. Biophys. Res. Commun. 101, 1131-1138. [23] Bikle, D.D., Pillai, S., Gee, E. and Hincenbergs, M. (1989) Endocrinology 124, 655-60. [24] E Merke, J., Milde, P., Lewicka, S., H:ugel, U., Klaus, G., Mangelsdorf, D.J., Haussler, M.R., Rauterberg, E.W. and Ritz, E. (1989). J. Clin. Invest. 83, 1903-15. [25] Binderup, L., Latini, S., Binderup, E., Bretting, C., Calverly, M. and Hansen, K. (1991) Biochem. Pharmacol. 42, 1569-1575. [26] Jassal, D., Han, R.N.N., Caniggia, I., Post, M. and Tanswell, A.K. (1991) In Vitro Cell Dev. Biol. 27, 625-632. [27] Dobbs, L.G., Gonzalez, R. and Williams, M.C. (1986) Am. Rev. Respir. Dis. 134, 141-145. [28] Edelson, J.D., Shannon, J.M. and Mason, R.J. (1988) Am. Rev. Respir. Dis. 138, 1268-1275. [29] Mason,, R.J., Walker, S.R., Shields, B.A., Henson, J.E. and Williams, M.C. (1985) Am. Rev. Respir. Dis. 131,786-788. [30] Vindelov, L.L., Christensen, I.J. and Nissen, N.I. (1983). Cytometry 3, 323-327. [31] Vindelov, L.L., Christensen, I.J., Keiding, N., Spang-Thomsen, M. and Nissen, N.I. (1983) Cytometry 3, 317-322. [32] Snedecor, G.W. and Cochran, W.G. (1980) Statistical methods. Iowa State University Press, Ames. [33] Steel, R.G.D. and Torrie, J.H. (1970) Principles and procedures of ststistics. McGraw Hill, New York. [34] Mitsuhashi, T., Morris, R.C. and Ives, H.E. (1991) J. Clin. Invest. 87, 1889-1895. [35] Reale, F.R. and Esterly, J.R. (1973) Pediatrics 51, 91-96.

[36] Morriss, F.H. and Boyd, R.D.H. (1988) in The Physiology of Reproduction (Knbil, E. and Neill, J., eds.), pp. 2043-2083, Raven Press. [37] Tanswell, K., Jassal, D., Post, M. and Edelson, J. (1992) Pediatr. Res. 31,324. [38] Kauffamn, S.L., Burri, P.H. and Weibel, E.R. (1974) Anat. Rec. 180, 63-76. [39] Rannels, S.R., Fischer, C.S., Heuser, L.J. and Rannels, D.E. (1987) Am. J. Physiol. 253, C759-C765. [40] Clement, A., Campisi, J., Farmer, S.R. and Brody, J.S. (1990) Proc. Natl. Acad. Sci. USA 87, 318-322. [41] Tryka, A.F., Witschi, H., Gossler, D.G., McArthur, A.H. and Clapp, N.K. (1986) Am. Rev. Respir. Dis. 133, 1055-1059. [42] Clement, A., Riedel, N. and Brody, J.S. (1990) Am. J. Respir. Cell. Mol. Biol. 3, 159-164. [43] Uhal, B.D. and Rannel, D.E. (1991) Am. J. Physiol. 261, L296L306. [44] Panos, R.J., Suwabe, A., Leslie, C.C. and Mason, R.J. (1990) Am. J. Respir. Cell. Mol. Biol. 3, 51-59. [45] Petkovich, P.M., Wrana, J.L., Grigoriadis, A.E., Heersche, J.N.M. and Sodek, J. (1987) J. Biol. Chem. 262, 13424-13428. [46] Taniguchi, K., Morimoto, S., Itoh, K., Morita, R., Fukuo, K., lmanaka, S. and Ogihara, T. (1990) Biochem. Int. 22, 37-44. [47] Martell, R.E., Strahler, J.R. and Simpson, R.U. (1992) J. Biol. Chem. 267, 7511-7519. [48] Scharla, S.H., Strong, D.D., Mohan, S., Baylink, D.J. and Linkart, T.A. (1991) Endocrinology 129, 3139-3146. [49] Moriwake, T., Tanaka, H., Kanzaki, S., Higuchi, J. and Seino, Y. (1992) Endocrinology 130, 1071-1073. [50] Shami, S.G., Evans, M.J. and Martinez,, L.A. (1986) Exp. Mol. Pathol. 44, 344-352. [51] Barbour, G.L., Coburn, J.W., Slatopolsky, E., Norman, A.W. and Horst, R.L. (1981) N. Engl. J. Med. 305, 440-443. [52] Reichel, H., Koeffler, H.P., Barbers, R. and Norman, A.W. (1987) J. Clin. Endocrinol. Metab. 65, 1201-1209. [53] Takizawa, H., Beckrnann, J.D., Shoji, S., Claasen, L.R., Ertl, R.F., Linder, J. and Rennard, S.I. (1990) Am. J. Respir. Cell. Mol. Biol. 2, 245-255.