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Dexamethasone Inhibits Growth Factor-Induced Type 3 Deiodinase Activity and mRNA Expression in a Cultured Cell Line Derived from Rat Neonatal Brown Fat Vascular-Stromal Cells

Departments of Medicine and Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756

Address all correspondence and requests for reprints to: Donald L. St. Germain, Departments of Medicine and Physiology, Dartmouth Medical School, One Medical Center Drive, Lebanon, New Hampshire 03756. E-mail: . stgermain{at}dartmouth.edu

	Abstract

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Studies examining the regulation of the type 3 deiodinase (D3) have been hampered by the lack of cell lines that constitutively express this enzyme. To address this issue, a new cell line, designated brown fat vascular-stromal (BVS-1), was generated by continuous subculturing of precursor cells derived from the vascular-stromal fraction of rat neonatal brown fat. BVS-1 cells did not differentiate into adipocytes when cultured for 5 d in DMEM supplemented with 2% newborn calf serum, 4 nM insulin, 2 nM T₃, and 10 nM dexamethasone (DEX). However, when cultured in regular medium, the cells expressed high levels of D3 activity (1–5 pmol/h per milligram protein) and mRNA. D3 mRNA was markedly induced by treatment for 6 h with epidermal growth factor, acid or basic fibroblast growth factors (10 ng/ml), or a 3-h treatment with a phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA), 1 µM] or 10% fetal bovine serum. However, preincubation of cells overnight with 50 nM DEX completely blocked the D3-inducing effects of basic fibroblast growth factor. The DEX effect was partially blocked when a glucocorticoid receptor antagonist was present. Overnight DEX treatment (50 nM) also decreased basal D3 activity by 80%. In summary, we have established BVS-1 cells as a continuous cell line useful for studying the regulation of D3 expression. Furthermore, we have shown that DEX inhibits growth factor-induced D3 expression in these cells.

	Introduction

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THE TYPE 3 deiodinase (D3), like the types 1 and 2 isoforms, belongs to a family of selenocysteine-containing enzymes that play a central role in thyroid hormone (TH) metabolism and thus the regulation of TH action in peripheral tissues (1). D3 inactivates TH because it deiodinates the inner ring of T₃ and T₄, and the resulting iodothyronines have no significant affinity for the nuclear TH receptor (2). D3 expression is very high in uterine decidual tissue (3), placenta (4, 5), and various fetal tissues (6). This expression pattern during pregnancy suggests that D3 is critical for protecting the early embryo and developing fetal tissues from the relatively high adult levels of TH. In the adult, modest D3 expression occurs in skin, the uterus, and the central nervous system (6, 7, 8).

In vivo studies have demonstrated that D3 activity in some tissues is regulated by TH status, being increased in hyperthyroidism and decreased in hypothyroidism (9). In addition, GH and dexamethasone (DEX) have been shown to decrease D3 mRNA transcription in the liver of chicken embryos (10). In certain primary rat cell culture models, such as differentiating brown preadipocytes and glial cells, we and others have described a pronounced up-regulation of D3 expression by the action of phorbol esters and various growth factors, e.g. epidermal growth factor (EGF) and acidic and basic fibroblast growth factors (aFGF and bFGF) (11, 12, 13). It is not known whether any of these growth factors regulate D3 in vivo, but their well-documented importance during implantation and embryonic development (14, 15) coincides with the high D3 activity found in decidual tissue, placenta, and fetal tissues. In addition, the hypothesis that bFGF up-regulates D3 in vivo comes from the known role of this growth factor in the support of angiogenesis (16), a process that is active in early embryogenesis. Further support for a relationship between D3 expression and angiogenesis comes from a recent report of Huang et al. (17) that describes high levels of D3 activity in hemangiomas from human infants. These findings suggest that growth factors are involved in D3 expression in vivo.

Despite the finding of D3 expression in primary culture of glial cells and brown preadipocytes, no established mammalian cell lines have been identified that express D3. In the present work, we have used precursor cells obtained from the vascular stroma of rat neonatal brown fat to establish a stable cell line that expresses basal and growth factor-induced D3. Using this model, we describe a novel effect of DEX to inhibit the induction of D3 expression by growth factors. This model system promises to be useful for studying the molecular events governing D3 expression.

	Materials and Methods

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Materials
DMEM, fetal bovine serum (FBS), BSA, agarose, and formamide were obtained from Life Technologies, Inc. (Gaithersburg, MD). Antibiotics, collagenase, bovine insulin, DEX, cycloheximide, guanidinium HCl, 3-N-morpholino-propanesulfonic acid, dithiothreitol, insulin, T₃, and retinoic acid (RA) were from Sigma (St. Louis, MO). Rosiglitazone (Avandia) was obtained from a local pharmacy, and the tablets were extracted with water before use. Growth factors were from R\|[amp ]\|D Systems (Minneapolis, MN). The aFGF was used in the presence of 50 µg/ml heparin (final concentration), as recommended for proper biological activity. Radiolabeled -[³²P] dCTP (3000 Ci/mmol) was from ICN (Costa Mesa, CA), and ¹²⁵I-T3 (2200 Ci/mmol) was obtained from NEN Life Science Products (Boston, MA). Nylon membranes (Nytran) were obtained from Schleicher \|[amp ]\| Schuell, Inc. (Keene, NH).

Culturing precursor cells from the vascular stroma of brown fat
Vascular stroma precursor cells were isolated from the interscapular brown adipose tissue (BAT) of 20-d-old rats (Sprague Dawley) according to the methods described by Néchad and colleagues (18, 19) with modifications (12), using collagenase digestion (0.2%) in DMEM+1.5% BSA at 37 C and filtration through 250-µm silk filters. Mature cells were allowed to float, and the infranatant was filtered through 25-µm silk filters and centrifuged. Cells were seeded in 25-cm² culture flasks at low density (400 cells/cm (2) and grown in DMEM supplemented with 10% FBS, glutamine (2 mM), and antibiotics (streptomycin and penicillin, 50 ng/ml). Culture medium was changed twice a week and cells were subcultured weekly at a 1:10 dilution. After 10 wk, the brown fat vascular-stromal (BVS-1) cells were passed three times a week and maintained in the same culture conditions except when used in experiments, as specified. BVS-1 cells were frozen in DMEM supplemented with 20% FBS and 10% dimethyl sulfoxide.

D3 activity
Cell cultures were washed with cold PBS; harvested with a buffer containing 0.25 sucrose, 1 mM dithiothreitol, 20 mM Tris-HCl, pH 7.4; and sonicated. Cell sonicates were assayed for D3 activity as described (3). The volume of the reaction mixture was 50 µl and contained between 1 and 10 µg of cell protein, 50 mM DTT, and 2 nM ¹²⁵I-T₃. A 1-h incubation time was used. Activity is expressed as femtomoles of T2 generated per hour and milligram of protein. Protein concentrations in all samples were determined using a protein assay (Bio-Rad Laboratories, Inc., Hercules, CA).

RNA preparation and Northern blot analysis
Total cellular RNA was extracted in guanidinium-HCl as described (13), using ethanol precipitation. For Northern analysis 15 µg total RNA was denatured and electrophoresed on a 2.2 M formaldehyde/1% agarose gel in 1x 3-N-morpholino-propanesulfonic acid buffer and transferred to nylon membranes as described (20). A 1100-bp fragment of a rat D3 cDNA clone (20), corresponding to most of the translated region of the D3 mRNA, was used as a probe by labeling with -[³²P]-dCTP using random primers (Pharmacia-Biotech, Piscataway, NJ). Filters were hybridized for 20–24 h at 42 C (in 50% formamide, 5x saline sodium citrate (SSC), 2x Denhardt’s, 0.1% SDS) and washed four times in 2xSSC/0.2% SDS at room temperature for 15 min and then twice in 0.1xSSC/0.2% SDS at 65 C for 20 min. Filters were autoradiographed and quantified by computer-assisted densitometry (Molecular Dynamics, Inc., Sunnyvale, CA). The filters were hybridized with cyclophilin as a control to correct for differences between lanes in the amount of RNA.

	Results

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Generation of the BVS-1 cell line
Precursor cells isolated from the interscapular brown fat of 20-d-old rats are able to briefly proliferate and differentiate into adipocytes when cultured under conditions that favor such a process (19). Such precursor cells account for less than 5% of the tissue cell population and originate from the vascular stroma (18, 19). The conversion of these cells to adipocytes results in the shortening of their viability in culture to just a few days. We have previously observed inducible expression of D3 in differentiating adipocytes (13), and our unpublished data suggest an association of D3 expression with the proliferative phase of the cultures, rather than with full-term mature adipocytes. Thus, to establish a cell culture model to study D3 regulation, we isolated and cultured rat brown fat precursor cells in conditions that favor proliferation rather than differentiation. Such conditions include low-density plating and the use of 10% FBS (instead of lower percentages of newborn and/or heat-inactivated serum) in the absence of insulin, T₃, or glucocorticoids. Under these conditions cells grew rapidly for the first 2 wk and then slower for the next 6 wk. However, by wk 10 the cells resumed rapid growth and had to be passed two or three times a week. Several aliquots of cells were frozen at passage 15. After being frozen in liquid nitrogen, at least 70% of the cells were viable upon replating, as determined by trypan blue exclusion. Before confluence, the cells showed a fibroblast-like morphology (Fig. 1), resembling that of the original precursors, although they had a somewhat larger cell body. A doubling time of 14 h was measured during the exponential phase of growth. According to their cells of origin, brown fat vascular stroma, we designated the cell line BVS-1 cells.

View larger version (139K): [in this window] [in a new window]   Figure 1. Photomicrograph of cultured BVS-1 cells (magnification x200).

A marked increase in lipoprotein lipase (LPL) expression has also been used as an early marker of adipose differentiation (22). We thus checked for LPL expression in BVS-1 cells and primary cultures before and after different treatments known to regulate its expression in vivo (22). BVS-1 cells did express detectable levels of LPL mRNA (Fig. 2B), but they were much lower than those found in primary cultures of stromal-vascular cells (Fig. 2A). A slight increase in LPL mRNA was observed in primary cultures when treated with norepinephrine. This up-regulation, which has been described as specific to brown fat (23), was not observed in BVS-1 cells. Because BVS-1 cells originated from the same stromal-vascular cells, these results suggest that there is only a short window of time in which stromal-vascular cells can differentiate to adipocytes and that BVS-1 cells have lost this capacity to undergo adipocyte conversion.

View larger version (32K): [in this window] [in a new window]   Figure 2. Northern analysis of LPL mRNA in brown fat precursor cells in primary culture (A) and BVS-1 cells (B) after treatment with different hormones known to regulate LPL expression in adipose tissue: T3, 5 nM x 24 h; I, insulin, 10 nM x 24 h; NE, norepinephrine, 1 µM x 6 h. A total of 15 µg total RNA were run per lane. The blot was also hybridized with a cyclophilin (Cy) probe to correct for the amount of RNA loaded. Films were exposed for 48 h (LPL) and 12 h (Cy).

View larger version (78K): [in this window] [in a new window]   Figure 3. Northern analysis of D3 mRNA in BVS-1 cells after treatment with various hormones and growth factors. A total of 15 µg total RNA were run per lane. Cells were maintained in 2% FBS supplemented medium for the last 20 h before harvesting. Doses and exposure times used were: EGF, aFGF, and bFGF all at 10 ng/ml x 6 h; DEX, 50 nM x 20 h; T3, 5 nM x 6 h; RA, 200 nM x 20 h; TPA, 1 µM x 3 h; and FBS, 10% x 3 h. The blot was also hybridized with a cyclophilin (Cy) probe to correct for the amount of RNA loaded. D3 and Cy signals were quantified and the ratio is represented in arbitrary units. Films were exposed for approximately 60 h (D3) and 12 h (Cy).

View larger version (68K): [in this window] [in a new window]   Figure 4. A, DEX inhibition of D3 mRNA induction. DEX at a dose of 50 nM was added to the cell culture media 24 h before harvesting. B, Dose and time dependence of the inhibitory effect of DEX are demonstrated. Cells were maintained with 2% FBS supplemented medium for the last 30 h before harvesting. A total of 15 µg total RNA were run per lane in both experiments. Doses and exposure times for aFGF, bFGF, TPA, and FBS were the same as in Fig. 3. Blots were hybridized with a cyclophilin (Cy) probe to correct for the amount of RNA loaded. Films were exposed approximately 60 h (D3) and 12 h (Cy).

We have previously shown that D3 mRNA expression in primary cultures of differentiating preadipocytes is transiently responsive to stimulation by bFGF at the time that the cells are starting to express adipocyte marker genes (13). Such induction requires protein synthesis and peaks at 6–8 h after bFGF addition (13). Induction of D3 mRNA expression by TPA and FBS peaks at significantly shorter times (2–3 h, Hernandez, A., and D. L. St. Germain, unpublished data). We thus determined whether protein synthesis was also needed for TPA- and FBS-dependent D3 induction in BVS-1 cells. The results demonstrate that cycloheximide also inhibits induction of D3 mRNA by these agents (Fig. 5A). Inhibition of transcription with actinomycin D also blocked D3 induction. Because induction of D3 mRNA under these circumstances is dependent on protein synthesis, it is not possible to determine whether the inhibitory effect exerted by DEX is mediated by a direct transcriptional effect through the glucocorticoid receptor or by induction of an inhibitory protein. However, treatment of cells with the antagonist RU486 prevents the DEX-induced inhibition of bFGF-dependent D3 mRNA induction (Fig. 5B, lanes 3 and 4), suggesting that the glucocorticoid receptor is required for this effect.

View larger version (40K): [in this window] [in a new window]   Figure 5. A, Transcription and protein synthesis dependence of D3 induction by TPA and serum. The same doses and exposure times for bFGF, TPA, and FBS were used as in Fig. 3. Actinomycin D (ActD, 5 µg/ml) and cycloheximide (CHX, 25 µM) were added 10 min before the agonists. B, The glucocorticoid receptor antagonist Ru486 blocks the inhibition of D3 mRNA by DEX. Ru486 (60 nM) and DEX (50 nM) were added to cells 20 h before harvesting. The bFGF (10 ng/ml) was added for the last 6 h. Cells were maintained with 2% FBS supplemented medium for the last 20 h. A total of 15 µg of total RNA were run per lane in both experiments. Blots were hybridized with a cyclophilin (Cy) probe to correct for the amount of RNA loaded. Films were exposed for approximately 60 h (D3) and 12 h (Cy).

View larger version (48K): [in this window] [in a new window]   Figure 6. Basal and induced D3 activity in BVS-1 cells after 18 passages (A) or 23 passages (B). Doses and exposure times used were: aFGF or bFGF, 10 ng/ml x 6 h; DEX, 50 nM x 20 h; TPA, 1 µM x 3 h; and FBS, 10% x 3 h. Results represent the means ± SD of triplicate culture dishes.

View larger version (37K): [in this window] [in a new window]   Figure 7. A, Basal and 3-h TPA-induced D3 activity in long-term BVS-1 cells after 42 passages. Cells were cultured for the last 2 wk in regular or differentiation medium (see Materials and Methods). B, Effect of selenium supplementation (50 nM for 4 d) on basal and TPA-induced D3 activity in BVS-1 cells after 45 passages. C, D3 activity half-life in regular and serum-free medium. CHX (25 µM) was added at time 0, and cells (passage 31) harvested at 4, 8.5, and 25 h. Cells not treated with CHX were harvested at 0 and 25 h as a control. All results represent the means ± SD of triplicate culture dishes.

Given that the D3 is a selenoprotein that requires the presence of the uncommon amino acid selenocysteine for translation, we determined how selenium supplementation of the culture medium affected D3 activity in the basal and the stimulated state. The results show that selenium supplementation (50 nM, 4 d) almost tripled basal D3 activity (Fig. 7B), but again no stimulation of activity by TPA was observed. To examine these phenomena further, we used cycloheximide to determine that the half-life of D3 activity was approximately 20 h and 33 h in the presence or absence of serum, respectively (Fig. 7C). These half-lives are much longer than those we previously measured in differentiating preadipocytes in primary culture [3 and 6 h, respectively, for the same conditions (12)].

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our understanding of the physiological role and regulation of D3 is limited, in part because of the lack of availability of appropriate model systems. In an attempt to establish a mammalian cell line that expresses this enzyme, we subcultured vascular stromal precursor cells from rat BAT under conditions that favor proliferation. The cell line was designated BVS-1 solely on the basis of its cellular origin (19). These cells express D3 mRNA and activity and appear suitable for studying the regulation of D3 by growth factors and other agents. Although these cells originate from the same brown fat vascular-stromal precursor cells that are capable of differentiating into adipocytes, BVS-1 cells have lost that capacity. It is possible that they have also lost some of the characteristics of the stromal-vascular cells from which they are derived. However, they have retained both high-level expression of D3 mRNA and responsiveness of this gene product to stimulation by EGF, aFGF, and bFGF. We also show that D3 expression is induced by TPA, as occurs in primary cultures of rat glial cells (11), and by serum. These findings are consistent with prior studies indicating that D3 activation occurs via the ERK signaling pathway and protein kinase C (27).

Using cultured BVS-1 cells, we report a novel effect of DEX, a glucocorticoid commonly used in adipocyte differentiating protocols. DEX is able to block completely the induction of D3 mRNA levels caused by growth factors, TPA, and serum. This effect is observed at doses as low as 25 nM and does not require preexposure to DEX before the cells are exposed to the growth factor. In fact, we have observed that addition of DEX after bFGF also inhibits D3 induction, although it does so only partially (data not shown). DEX also decreases basal D3 mRNA levels in these cells, an observation consistent with that previously described in a different model system, the developing chicken liver (10). Because induction of D3 mRNA is dependent on protein synthesis in BVS-1 cells, it is not possible to determine whether the effect of DEX is due to direct transcriptional repression of the D3 gene or is a secondary effect.

The inhibitory effect of DEX on the induction of D3 by growth factors may have therapeutic relevance to patients with infantile hemangiomas. These vascular tumors, which are known to produce high levels of bFGF and other angiogenic factors (28), have recently been reported to express high levels of D3 activity (17) that may result in hypothyroidism. To the extent that D3 in such tumors is being stimulated by growth factors, treatment with glucocorticoids may decrease D3 activity and improve thyroid status.

Considering the origin of this cell line and the known ability of DEX to promote adipocyte conversion, these data suggest that D3 expression is linked to the proliferation of precursor cells rather than to full-term mature adipocytes. This finding is consistent with the observation that brown fat harvested from rodents does not contain any demonstrable D3 activity (13) and that these precursor cells account for a very small percentage of the BAT cell population (19). Thus, D3 expression may be important in regulating the number of precursor cells that differentiate into functional brown adipocytes, a process that requires TH (29). This role for D3 may be applicable to other cell differentiation events that rely on TH.

Our finding that a glucocorticoid receptor antagonist prevents most of the DEX-induced inhibition suggests that the interaction of DEX with the glucocorticoid receptor is needed for this effect. No glucocorticoid response elements have been characterized in the D3 gene promoter, but we have identified an enhancer located 3' of the D3 gene that is conserved in mouse and human (Hernandez, A., and D. L. St. Germain, unpublished observations). This enhancer contains putative activator protein-1 (AP-1) and serum response elements that may mediate part of the D3 gene activation by growth factors. Transient, rapid induction and synthesis of the c-jun and c-fos oncoproteins are needed for the activation of AP-1 sites (30), and this might explain the dependence on protein synthesis of the D3 induction by growth factors. On the other hand, a potential explanation for the inhibitory effect of DEX is its known antagonism of AP-1 elements (31). Also, the glucocorticoid receptor can antagonize serum response elements, as reported in the c-fos gene (32).

A surprising finding in this model is that BVS-1 cells behave differently from the precursor cells with regard to D3 activity. In the latter, basal D3 activity is relatively low but is markedly increased by treatment with growth factors (12). However, in BVS-1 cells, basal D3 activity is very high during early passages and modest or no increases are found with growth factor treatment, despite significant increases in D3 mRNA levels. After numerous passages, BVS-1 cells show somewhat lesser levels of basal D3 expression and limited stimulation of activity by TPA treatment is observed.

There are several possible explanations for the discrepancy between the levels of induction by growth factors of D3 activity and mRNA in BSV-1 cells. One possibility is that the D3 mRNA detected by Northern analysis is defective. This is unlikely given that the size of the mRNA detected is that expected from previous studies (33). In addition, experiments using quantitative RT-PCR and Northern analysis with partial D3 cDNAs as probes demonstrate that the entire coding and 5' and 3' untranslated regions are present in the BSV-1 message (data not shown). An alternative explanation, which cannot be excluded at the present time, is that the BSV-1 cells lack a factor(s) required for efficient D3 mRNA translation or may express a factor(s) that interacts with the D3 mRNA and decrease its availability for translation. Indeed, selenium supplementation did increase the level of basal D3 activity but did not allow for significant stimulation by growth factors.

The most likely explanation for the difference in D3 mRNA and activity comes from the observations that: 1) BVS-1 cells have a very high basal level of D3 activity (10- to 20-fold higher than in precursor cells) that appears to be due in part to a relatively long half-life of the protein (8 to 10 times longer than that found in precursor cells) as judged by the stability of D3 activity when cells are cultured in the presence of cycloheximide and 2) stimulation of D3 mRNA by growth factors is transient with levels returning to baseline at 6 h after induction by FBS or TPA (data not shown). Thus, the short-lived peak of growth factor-induced D3 mRNA likely results in relatively little new enzyme protein, compared with that already present. This explanation is supported by the observation that in BSV-1 cells in long-term culture, in which D3 activity levels have decreased by 80%, TPA induction does result in a significant rise in activity levels.

In summary, using BVS-1 cells, a new cell line derived from vascular stromal precursor cells of brown fat, we have described a novel effect of DEX in inhibiting the induction of D3 by growth factors. These cells should provide a useful model system for studying the molecular mechanisms by which growth factors and other compounds regulate D3 expression. Such studies may provide information regarding the physiological role of D3 during development.

	Footnotes

 
This work was supported by NIH Grants DK-54716 and DK-42271.

Abbreviations: aFGF, Acidic fibroblast growth factor; AP-1, activator protein-1; BAT, brown adipose tissue; bFGF, basic fibroblast growth factor; BVS-1, brown fat vascular-stromal cell line; D3, type 3 deiodinase; DEX, dexamethasone; EGF, epidermal growth factor; FBS, fetal bovine serum; LPL, lipoprotein lipase; RA, retinoic acid; SSC, saline sodium citrate; TH, thyroid hormone; TPA, 12-O-tetradecanoylphorbol-13-acetate.

Received December 13, 2001.

Accepted for publication April 2, 2002.

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				S Van der Geyten and V M Darras Developmentally defined regulation of thyroid hormone metabolism by glucocorticoids in the rat J. Endocrinol., May 1, 2005; 185(2): 327 - 336. [Abstract] [Full Text] [PDF]

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