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Laboratories of Molecular Hypertension (P.S.B., F.H.S., K.K., Z.K.) and Morphology (R.R.D., R.D), Baker Medical Research Institute, Melbourne, Victoria 8008, Australia
Address all correspondence and requests for reprints to: Dr. Z. Krozowski, Molecular Hypertension Laboratory, Baker Medical Research Institute, P.O. Box 6492, Melbourne, Victoria 8008, Australia. E-mail: zygmunt.krozowski{at}baker.edu.au
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To gain further insight into the role of 11ßHSD1, it is important to define the cellular distribution of the enzyme in tissues of an animal model. Although early work in the rat showed the tissue distribution by Western blot analysis, there are limited immunohistochemical reports in this species, and the intensity of staining is at times at odds with the levels of activity observed (8, 9). An antibody has been generated against the N-terminus of human 11ßHSD1, and immunolocalization has been performed on a wide range of tissues (10). Comparison of those results with enzymatic studies in the rat suggest important species differences. In this study we examined 11ßHSD1 distribution in a range of rat tissues using a novel immunopurified polyclonal antibody directed against peptides corresponding to the N- and C-termini of 11ßHSD1. Western blot analysis confirmed the presence of an antigen of the correct size and intensity consistent with levels of enzyme activity previously reported. Transmission microscopy was also used to identify cell types expressing 11ßHSD1 in the lung and kidney. The results show that in many tissues 11ßHSD1 is present in isolated cells, consistent with a paracrine role for the enzyme.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunohistochemistry
Two adult male Sprague Dawley rats were used. Tissues of
anesthetized rats were perfusion fixed (retrograde aorta) with 4%
paraformaldehyde plus 0.05% glutaraldehyde and 4% sucrose in PBS.
Five-micron thick cross-sections of paraffin-embedded tissue were cut and mounted on Vectabond adhesive-coated slides (Vector Laboratories, Inc., Burlingame, CA). Sections were dewaxed in xylene and rehydrated in ethanol to water, then washed in PBS, pH 7.4, and treated with 0.3% H2O2 in PBS to block endogenous peroxidase activity. After an additional wash in PBS, sections were incubated with 10% horse serum in PBS for 30 min before application of the primary antibody, RAH113, at 1–2 µg/ml for 1 h. Control sections were incubated with antibody diluent solution (PBS-2% horse serum) or an irrelevant antibody in place of primary antibody. After 1 h at room temperature the sections were washed in PBS. Tissue-bound primary antibody was detected using biotinylated secondary antibody and the avidin-biotin peroxidase complex method (Vector Laboratories, Inc.) with diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) as the chromogen. Sections were counterstained with hematoxylin, dehydrated in alcohol, and mounted in Depex mounting medium (BDH, Poole, UK).
Western blot analysis
Each tissue (
200 mg) was homogenized in buffer [10
mM phosphate (pH 7.5), 0.14 M NaCl, and 0.25
M sucrose] containing a cocktail of protease inhibitors
(174 µg/ml phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, 1
µg/ml leupeptin, and 1 µg/ml pepstatin A). Protein concentrations
were determined by the Bradford method (Bio-Rad Laboratories, Inc., Richmond, CA). Denaturing SDS-PAGE was performed on a
12.5% polyacrylamide gel. Each well was loaded with 100 µg protein,
with the exception of the liver (20 µg). Prestained mol wt standards
were obtained from Life Technologies, Inc. (Grand Island,
NY). Proteins were transferred onto Protran nitrocellulose membranes
(0.45 µm; Schleicher & Schuell, Inc., Darmstadt,
Germany) at 4 C for 2.5 h. The membrane was blocked with
5% skim milk in PBS-0.1% Tween 20 (PBST) for 1.5 h, and then
incubated overnight at 4 C with the primary antibody RAH113 (1.5
µg/ml) in PBST. The following day the membrane was washed with PBST
(four 15-min washes) at room temperature and incubated for 1.5 h
with a sheep antirabbit IgG secondary antibody (Amrad, Melbourne,
Australia) at a dilution of 1:1000 in PBST. The membrane was again
washed as described above, and detection was performed via a
chemiluminescent kit (NEN Life Science Products, Boston,
MA).
Electron microscopy
Organs of interest were immediately dissected into 1-mm cubes
and placed into fresh fixative overnight at 4 C. For morphology
studies, tissue was washed in PBS buffer, postfixed 1 h at room
temperature in 1% osmium tetroxide, dehydrated through a graded series
of acetones, infiltrated with, and embedded in Procure 812 (Epon
substitute, ProSci Tech, Thuringowa Central, Queensland, Australia).
Ultrathin sections were contrasted with 10% uranyl acetate in 75%
methanol for 5 min and with lead citrate for 5 min.
For immunogold labeling, tissues were dissected further into blocks of 0.5 mm, and aldehydes were blocked in 0.05 M glycine in HEPES-buffered saline (HBS) for 1 h. The samples were then cryoprotected in 15% polyvinylpyrrolidone containing 1.7 M sucrose overnight at 4 C, hardened in liquid nitrogen, freeze-substituted in 0.5% uranyl acetate and 1% glutaraldehyde in dry methanol at -90 C for 70 h, and rinsed 1 h in dry methanol. The temperature was increased by 5 C/h to -45 C, and tissues were infiltrated with Lowicryl HM20 (Polysciences Inc., Warrington, PA) and UV-polymerized for 48 h. Blocks were raised to room temperature and sectioned (70 nm) onto Formvar-coated nickel grids. For immunolabeling, grids were floated for 30 min on 2% gelatin in 0.1 M phosphate buffer. Nonspecific binding was blocked by incubation for 15 min in blocking solution (0.2% ovalbumin, 0.2% fish skin gelatin, and 0.24% glycine in PBS). Primary antibody was diluted in blocking solution to 2 µg/ml, and sections were incubated 30 min at room temperature and then washed twice for 5 min each time in PBS, followed by 5 min in 20 mM Tris, 20 mM NaN3, 225 mM NaCl, and 0.1% ovalbumin, pH 8.2 (secondary buffer). Grids were incubated for 30 min at room temperature on a goat F(ab)2 antirabbit 10-nm gold probe (EM.GFAR10, British Biocell International, Cardiff, UK) diluted 1:50 in secondary antibody buffer, washed three times for 5 min each time on secondary buffer, then rinsed with HBS and postfixed for 5 min in 2.5% glutaraldehyde in HBS. After several washes in water from Milli-Q Water Purification System (Millipore, MA), grids were air-dried. Before examination, sections were contrasted with 3% uranyl acetate for 5 min and with lead citrate for 3 min. Control sections for lung and kidney were treated with preimmune serum (2 µg/ml) as the primary antibody.
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Results |
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. In the liver
(Fig. 1A) staining was concentrated around the central vein and was
absent in the vicinity of bile ducts. There was no staining in the
liver when the antibody was incubated with nonimmune serum (results not
shown). The lung (Fig. 1B) showed staining in alveoli, whereas the
stomach (Fig. 1C) expressed 11ßHSD1 in parietal cells. In the kidney
cortex (Fig. 1D) 11ßHSD1 was present in the proximal tubule, but in
the renal medulla and papilla staining was found in interstitial cells
(Fig. 1, E and F). The renal distribution of 11ßHSD1 contrasted with
that of 11ßHSD2, which in comparable sections was localized to the
distal convoluted tubule in the cortex (Fig. 1G), and to collecting
ducts in the medulla (Fig. 1H) and papilla (Fig. 1I).
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). In the adrenal cortex 11ßHSD1 was present in the outer layer of
cells corresponding to the glomerulosa, but was absent from the
fasciculata/reticularis (Fig. 1K). Staining for 11ßHSD1 in the
adrenal glomerulosa was to long thin structures that may be projections
of interstitial fibroblasts or neuronal in origin and did not appear to
be to aldosterone-secreting cells, which have a columnar or pyramidal
shape. 11ßHSD1 was also evident in the adrenal medulla (Fig. 1L). In
contrast, 11ßHSD2 was seen in the fasciculata/reticularis, but was
absent from the glomerulosa and medulla (Fig. 1M). On higher
magnification, 11ßHSD2 staining was evident in cord-like structures,
consistent with the presence of 11ßHSD2 in steroid-secreting cells.
In the heart 11ßHSD1 was present in cells resembling interstitial
fibroblasts in the endocardium, but not in myocytes (Fig. 1N). The only
cells staining in the myocardial layer were associated with blood
vessels and appeared to be adventitial fibroblasts (Fig. 1O).
Western blot analysis was also performed on all rat tissues studied by
immunohistochemistry (Fig. 2). A band
migrating at 34 kDa was evident in all tissues in which 11ßHSD1 was
observed, and the intensity of this band correlated to the amount of
immunostaining. The only exception was the pancreas, where a weak
signal was seen on the Western blot at 34 kDa, whereas intense
immunostaining was obtained in acini. An intense band seen at 25 kDa
may account for the acinar staining, but the relationship of this
epitope to 11ßHSD1 is unknown. A weak band was also seen at 35 kDa in
antral and fundic stomach and in the pancreas. The brain and testis
also showed a 34-kDa band, and the intensities of the bands are
consistent with the amounts of activity previously reported in these
tissues (8). The only tissue in which 11ßHSD1 appeared
to be below the limits of detection was the antral stomach, consistent
with our inability to detect immunostaining in this organ.
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, A
and B, shows that it is the interstitial fibroblast that expresses
11ßHSD1 in the lung. About 10-fold less expression was seen in type
II pneumocytes (results not shown). Immunogold labeling was seen mainly
on the rough endoplasmic reticulum and the nuclear envelope (Fig. 3, B
and C). Morphological studies also identified interstitial cells in the
renal papilla as the cell type containing 11ßHSD1 (Fig. 4, A and B). Immunogold labeling was
again seen primarily along the rough endoplasmic reticulum and nuclear
membrane (Fig. 4C). Control lung and kidney sections treated with
dilute preimmune serum did not show evidence of labeling.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study we observed a concentration of 11ßHSD1-positive cells around the central hepatic vein, identical with the pattern observed in human liver (10). A number of scenarios could explain this cellular distribution. A lineage model has been proposed for hepatocytes, starting with stem cell precursors in the portal zone and ending with aging cells around the central vein (16). 11ßHSD1 could be induced in hepatocytes as they progress toward the end of the lineage. Alternately, the pattern of distribution may be the result of zonation associated with a metabolic pathway (17). Although the major species migrated at 34 kDa on Western blots, a minor band was evident at approximately 29 kDa. This species was also evident in all other tissues expressing elevated levels of 11ßHSD1 and may be the result of translation starting from the second ATG codon. This truncated protein was previously named 11ßHSD1B and has been shown to be inactive (18, 19).
Glucocorticoids play an important role in the development and maintenance of normal lung function. Both active glucocorticoids and their 11-keto metabolites increase the production of surfactant precursor phosphatidylcholine from pneumocytes (20). Glucocorticoids also modulate the morphology of the lung by increasing alveolar wall thinning and the frequency of epithelial-interstitial cell contacts (21). Pulmonary 11ßHSD1 has 4-(methylnitrosammino)-1-(3-pyridyl)- 1-butanone carbonyl reductase activity and plays a role in the detoxification of inhaled compounds present in cigarette smoke. A 20-fold variation in enzyme levels was seen in subjects, suggesting that lower levels may predispose to disease (22). Electron microscopy showed that interstitial fibroblasts contained the highest levels of 11ßHSD1 in the lung, with markedly lower levels in the type II pneumocyte. This suggests that fibroblasts may act as paracrine regulators of glucocorticoid activity in the lung. Immunogold labeling was evident mainly in the endoplasmic reticulum, but also on the nuclear membrane. As glucocorticoids are known to exert their effects within the nucleus, nuclear 11ßHSD1 is ideally placed to modulate glucocorticoid action. Studies on 11ßHSD2 have also provided evidence for a nuclear localization in some tissues. It has been suggested that tissue-specific factors, such as the presence of mineralocorticoid receptor, may determine such a distribution (23).
The presence of 11ßHSD1 in the proximal tubules of the kidney is well documented, but our detection of the enzyme in renal interstitial cells of the medulla and papilla was an unexpected finding. Ultrastructurally, these cells can be seen to contain lipid-containing secretory vesicles. Renal interstitial cells have been extensively studied and shown to secrete the hypotensive agent medullipin I, contain receptors for a number of vasoactive substances, and produce extracellular matrix (24, 25). 11ßHSD1 could thus modulate renal function by regulating synthesis of secretory products or affecting receptor synthesis. It is ideally sited in renal interstitial cells to use the 11-keto glucocorticoid metabolites produced by 11ßHSD2 in adjacent renal tubules. 11ßHSD1 is highly expressed in rodents and sheep, but is present at barely detectable levels in the human kidney (26, 27). These differences may reflect utilization of different enzymes for the inactivation of xenobiotics (28).
In a previous study pancreas showed 11ßHSD1 activity, but there was no evidence for the protein by Western blots, leading investigators to conclude that another isoform may be responsible for the activity (29). However, in the present study we were able to demonstrate clear evidence for the 34-kDa 11ßHSD1 pancreatic protein. A band of high intensity was also found at 25 kDa. Given the discreet nature of the band at 34 kDa, it would appear that the 25-kDa band is not a breakdown product of 11ßHSD1. It has previously been shown that transcripts from an alternative promoter give rise to an inactive, N-terminally truncated protein (18, 19). We have shown that the 11ßHSD1B protein migrates at 26 kDa (18), consistent with the 25-kDa band we observed in the present study. Thus, the strong acinar staining could be due to 11ßHSD1B, the biological function of which remains unknown. It is probable that the majority of immunostaining evident in the pancreas is not due to 11ßHSD1B enzyme and that 11ßHSD1 is present at lower levels than suggested by our immunostaining results. This would be consistent with the low levels of activity detected in this tissue and the weak signal seen at 34 kDa on the Western blot. Pancreatic 11ßHSD1 could contribute to glucocorticoid modulation of pancreatic amylase activity (30). We also detected 11ßHSD1 immunostaining in the parietal cells of the fundus of the stomach and observed the appropriate band on Western blots, whereas there was no evidence for 11ßHSD1 by either technique in antral stomach. This observation contrasts with our previous work in the human, where 11ßHSD2 and mineralocorticoid receptor were found in parietal cells (31).
In the adrenal gland 11ßHSD1 was observed in the glomerulosa and medulla, whereas 11ßHSD2 was present only in the fasciculata/reticularis, with staining more intense toward the medulla. The 11ßHSD1 staining appeared to resemble a pattern not unlike that expected for neuronal cells. In previous adrenal studies 11ßHSD1 was localized by in situ hybridization to the cortico-medullary junction, where it is thought to modulate the activity of phenylethanolamine N-methyltransferase that catalyzes the conversion of noradrenaline to adrenaline (32). The glucocorticoid receptor-deficient mouse does not synthesize adrenaline and lacks a central adrenal medulla, implying the existence of a glucocorticoid-dependent cell population consistent with our localization of 11ßHSD1 (33). In man 11ßHSD1 is present in all three zones of the adrenal cortex (10), whereas 11ßHSD2 appears to be absent from normal adult adrenals. However, it is present in the human fetal adrenal and in steroid-synthesizing cells of adrenal carcinoma and adenoma (34).
We also investigated 11ßHSD1 in the testis and brain by Western blot analysis, because some studies have provided evidence inconsistent with the presence of classical 11ßHSD1 protein. Two studies show that the direction of the enzyme reaction was dependent on the composition of the incubation medium and that this phenomenon was limited to Leydig cells (35, 36). Our results show the presence of high amounts of the 34-kDa species in the testis confirming the presence of the classical 11ßHSD1 enzyme. Other studies demonstrated a different steroid specificity in the brain from that in the liver, and that the brain activity was more stable (29). A 26-kDa species was detected with one of the antibodies instead of the 34-kDa 11ßHSD1, suggesting that there may be neuron-specific differences at the molecular level. In our study RAH113 detected only a 34-kDa species in the brain. To rule out the possibility of differential splicing we cloned and sequenced the brain 11ßHSD1 cDNA. We identified an additional amino acid residue when comparing the sequence to that deposited in the databases (239LEIK242 should be 239LEIIK243), but sequencing of the original liver clone (provided by Dr. Perry White) (37) also showed the extra residue (results not shown). This correction to the sequence now makes the rat, human, mouse, and sheep 11ßHSD1 proteins identical in this region. Thus, no differences were apparent between the rat liver and brain 11ßHSD1 proteins that would account for the differences in enzymatic properties. A possible explanation may be the association of neuronal proteins with 11ßHSD1 to engender conformational changes.
In the heart 11ßHSD1 was restricted to the interstitial cells of the endocardium and isolated cells surrounding cardiac vessels. The number of 11ßHSD1-positive cells is consistent with the low signal on the Western blot in the present study and the low amounts of 11ßHSD1 activity previously reported in this tissue (9). The endocardium consists of endothelial cells, smooth muscle cells, collagen, and fibroblasts. 11ßHSD1 has been demonstrated in isolated rat cardiac fibroblasts, but not endothelial cells, suggesting that the staining in the present study may be to fibroblasts and consistent with our observations in the lung and kidney (38). The role of glucocorticoid-modulating enzymes in the heart seems complex, as gender- and strain-specific differences in 11ßHSD1 activity have been observed in the rat (39). Additionally, in the human heart, enzyme activity is NAD, but not NADP, dependent (40). Staining in the vasculature is consistent with evidence that 11ßHSD1 increases glucocorticoid within the vessel wall (41) and potentiates the activity of adrenergic agonists.
In mice harboring a deletion of the 11ßHSD1 gene there is clear evidence for a role of 11ßHSD1 in maintaining circulating glucocorticoid levels given the presence of compensatory adrenal hyperplasia. Our results also provide evidence for an intracrine and paracrine role for this enzyme in many tissues. The availability of a characterized, specific rat 11ßHSD1 antibody should facilitate further studies into the physiological role of this enzyme.
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Acknowledgments |
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Footnotes |
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2 Current address: First Department of Surgery, Tohoku University
School of Medicine, Sendai 980, Japan.
Received September 12, 2000.
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References |
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cortisol conversion in subjects with central adiposity.
J Clin Endocrinol Metab 84:1022–1027
-hydroxycholesterol dehydrogenase. Similarity to type I
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