1 Department of Molecular Pathology, Institute of Pathology and 2 Institute of Physiology, University of Tübingen, D-72076, Tübingen, Germany
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ABSTRACT |
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Transcript levels of the human serine/threonine kinase h-sgk have been found to be highest in pancreas. In the present study, localization and regulation of h-sgk transcription in pancreatic tissue were elucidated. As was apparent from radioactive in situ hybridization, most pancreatic acinar cells expressed high levels of h-sgk mRNA. h-sgk mRNA-positive cells were also found in ductal epithelia but not in pancreatic islets. In biopsy specimens from patients with pancreatitis, h-sgk mRNA levels were decreased in acinar cells but abundant in numerous mononuclear interstitial cells within areas of pancreatic necrosis and fibrosis. As shown by Northern blotting, h-sgk transcription in DAN-G pancreatic tumor cells is upregulated by osmotic cell shrinkage, serum, phorbol esters (phorbol 12,13-didecanoate), and Ca2+ ionophore A-23187 and decreased by staurosporine and cAMP. In conclusion, h-sgk transcription is regulated not only by cell volume but also by serum, protein kinase C stimulation, cAMP, and increase of intracellular Ca2+ activity. The kinase may participate not only in normal function of exocrine pancreas but also in fibrosing pancreatitis.
serine/threonine kinase; acinar cells; pancreatitis; pancreatic ducts; macrophages
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INTRODUCTION |
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ONE OF THE IMPORTANT CHALLENGES of cell volume constancy is epithelial transport. Cellular uptake and extrusion of transported ions and organic substances must be coordinated to avoid excessive alterations of cell volume. Similar to other epithelia, pancreatic cells regulate their cell volume by activation of ion transport across the cell membrane (11). The mechanisms linking cell volume to volume regulatory ion transport, however, remain poorly understood. Recently, the putative serine/threonine kinase h-sgk was cloned from Hep G2 cells and shown to be transcriptionally regulated by cell volume (17). The kinase is highly homologous to the serum glucocorticoid-inducible kinase sgk previously cloned from rat mammary tumor cells (20). Expression of h-sgk is markedly increased on hypertonic and isotonic cell shrinkage and is decreased on hypotonic and isotonic cell swelling (17). As shown by Northern blot analysis, the kinase is expressed in all tissues studied, including pancreas, liver, heart, skeletal muscle, placenta, kidney, and brain (17). However, by far the highest transcript levels were observed in the pancreas, pointing to an important role of h-sgk in this tissue (17). Because epithelial transport involves several cell volume regulatory mechanisms (9), the cell volume-sensitive kinase h-sgk, if expressed, could influence the function of the exocrine pancreas. The present study was performed to elucidate the cellular distribution of h-sgk mRNA in normal and inflamed pancreatic tissue. Additional experiments were performed to disclose transcriptional regulation of h-sgk in pancreatic tumor cells.
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MATERIALS AND METHODS |
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In situ hybridization. Tissue specimens of normal pancreas (n = 6) and of pancreas from patients with acute (n = 6) and chronic (n = 6) pancreatitis obtained at routine biopsies were fixed in 4% paraformaldehyde-0.1 M sodium phosphate buffer (pH 7.2) for 4 h and embedded in paraffin. Four-micrometer tissue sections were dewaxed and hybridized basically as described previously (5-7). The hybridization mixture contained either the 35S-labeled RNA antisense or sense control h-sgk probe (500 ng/ml) in 10 mM Tris · HCl, pH 7.4, 50% (vol/vol) deionized formamide, 600 mM NaCl, 1 mM EDTA, 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.05% bovine serum albumin, 10% dextran sulfate, 10 mM dithiothreitol, 200 µg/ml denatured sonicated salmon sperm DNA, and 100 µg/ml rabbit liver tRNA. Hybridization with RNA probes proceeded at 42°C for 18 h. Slides were then washed as described previously (5, 7) followed by 1 h at 55°C in 2× standard saline citrate. Nonhybridized single-stranded RNA probes were digested by RNase A (20 µg/ml) in 10 mM Tris · HCl, pH 8.0, and 0.5 M NaCl for 30 min at 37°C. Tissue slide preparations were autoradiographed for 3 wk (7) and stained with hematoxylin and eosin.
Transcriptional regulation of h-sgk by cell volume. DAN-G cells (pancreas carcinoma cells, ACC 249) were maintained in RPMI 1640 (GIBCO BRL), 5% CO2, and 10 mM glucose at 37°C, pH 7.4, supplemented with 10% (vol/vol) FCS. Cells were grown to 90% confluence and subsequently homogenized in TRIzol (GIBCO BRL) (~0.4 × 106 cells/sample). Total RNA was isolated as indicated in the protocol provided by the distributor. Northern blots were prepared with 15 or 20 µg of total RNA, each with a separate control that had been electrophoresed through 10 g/l agarose gels in the presence of 2.4 M formaldehyde. Vacuum blotting (Appligene Oncor Trans DNA express vacuum blotter, Appligene, Heidelberg, Germany) was used for transfer of the RNA on positively charged nylon membranes (Boehringer Mannheim, Mannheim, Germany), which were then cross-linked under ultraviolet light (UV Stratalinker 2400, Stratagene, Heidelberg, Germany). Overnight hybridization was performed in DIG-Easy-Hyb (Boehringer Mannheim) at a probe concentration of 25 µg/l at 50°C. The digoxigenin-labeled probe was generated by PCR as described in detail previously (17). For autoradiography, filters were exposed for an average of 5 min to X-ray film (Kodak).
Fluorescence measurements for determination of cell volume. DAN-G cells were grown on glass coverslips as described in Transcriptional regulation of h-sgk by cell volume. Calcein fluorescence intensity was utilized for determination of cell volume changes of single DAN-G cells. To this end, cells were loaded by exposure to 2 µM calcein AM (Molecular Probes) for 15 min and cell volume was measured microfluorometrically by exciting the dye with a 10-µm-diameter spot of light at 497 nm while monitoring the emission at 512 nm. Calcein is insensitive to changes in intracellular pH and Ca2+ (1). Swelling or shrinkage of cells is accompanied by a decrease or increase, respectively, in dye concentration in the cells (14). Therefore, changes in cell volume are expected to be reflected by changes in the fluorescence intensity, i.e., decreased intensity during cell swelling and increased intensity during cell shrinkage. A linear relationship between osmolarity of the perfusion medium and fluorescence intensity has been demonstrated using this technique (1). The relationship is linear because dye concentration is inversely proportional to cell volume and cell volume is inversely proportional to external osmolarity for cells that exhibit osmometric behavior. Fluorescence was measured using a ×100 oil-immersion lens (Zeiss). Fluorescence in the absence of calcein was <1% of the values in the presence of the dye and was not significantly modified by the experimental maneuvers. Measurements were performed at 37°C. The control solution contained (in mM) 114 NaCl, 21 NaHCO3, 5 KCl, 5 glucose, 1.2 CaCl2, 1 Na2HPO4, and 0.8 MgCl2, pH 7.4, equilibrated with 95% O2-5% CO2. To increase osmolarity, 50 mM NaCl or 100 mM raffinose was added to the solution.
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RESULTS |
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Localization of h-sgk mRNA in normal pancreas.
To elucidate the mRNA distribution patterns of h-sgk kinase in
pancreatic tissue at the cellular level, various tissue specimens of
intact pancreas were investigated by in situ hybridization. As
demonstrated in Fig. 1A,
hybridization of representative pancreatic specimens with the
35S-labeled h-sgk-specific antisense mRNA probe showed a
widespread expression pattern of h-sgk kinase mRNA in the exocrine
pancreas. Importantly, a considerable variation of levels of
transcription was observed in the numerous hybridization-positive
acinar cells. In addition to cells of the acini, cells of the
pancreatic ductal system were also found to express h-sgk mRNA in
differing amounts (Fig. 1B). Moreover, a small number of
h-sgk mRNA-positive mononuclear cells were scattered within the
connective tissue around the pancreatic ducts (Fig. 1B). No
expression of h-sgk mRNA was observed in pancreatic islets.
Hybridization with the 35S-labeled sense mRNA for control
did not lead to any labeling of pancreatic cells (Fig. 1C).
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h-sgk mRNA distribution in pancreatitis.
To compare h-sgk transcription patterns of inflamed pancreas to normal
tissue, biopsy material was investigated by radioactive in situ
hybridization. Figure 2A
represents an example of typical hybridization patterns of pancreatic
tissue specimens that were obtained from patients with acute
pancreatitis. As demonstrated in Fig. 2A, transcription of
h-sgk mRNA in acinar cells of affected pancreas is markedly reduced
compared with intact tissue. However, h-sgk mRNA was consistently
observed in interstitial cells that were interspersed between the
acini. In addition, investigation of tissue probes from patients with
chronic pancreatitis revealed abundant hybridization-positive
mononuclear cells in areas of fibrosis (Fig. 2B).
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Regulation of transcript levels in DAN-G pancreatic cells.
As demonstrated by Northern blot analysis, DAN-G cells transcribe h-sgk
depending on ambient osmolarity (Fig. 3).
Within 2 h, the transcript levels are enhanced by an increase of
extracellular osmolarity caused by addition of either 50 mM NaCl
[52 ± 19% (SE), n = 7] or 100 mM raffinose
(25 ± 9%, n = 3). Moreover, addition of 10% FCS
(59 ± 18%, n = 4), 10 µM Ca2+
ionophore A-23187 (128 ± 61%, n = 4), and 100 nM
phorbol 12,13-didecanoate (PDD; 108 ± 26%, n = 9) significantly increased h-sgk transcript levels, whereas 1 µM
staurosporine (63 ± 8%, n = 5) and 1 mM dibutyryl cAMP (
29.2 ± 9%, n = 7)
significantly decreased h-sgk transcript levels (Fig. 3). In cells
pretreated for 2 h with the protein kinase C inhibitor calphostin
C (1 µM), neither 100 nM PDD (17 ± 11%, n = 3)
nor 50 mM NaCl (10 ± 4%, n = 3) significantly increased h-sgk transcript levels. The values were significantly different from the respective effects in the absence of calphostin C.
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Cell volume measurements.
An increase of extracellular NaCl concentration by 50 mM increased
calcein fluorescence by 8.6 ± 1.6% (n = 4),
reflecting osmotic cell shrinkage (Fig.
4). A similar increase of calcein fluorescence was observed with addition of 100 mM raffinose (8.2 ± 1.6%, n = 6), calcium ionophore 10 µM A-23187
(7.4 ± 1.7%, n = 5), and 1 mM dibutyryl cAMP
(5.2 ± 0.6%, n = 6). PDD (100 nM) did not
significantly increase calcein fluorescence (0.4 ± 0.7%, n = 5).
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DISCUSSION |
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The present data demonstrate that h-sgk mRNA is differentially expressed in distinct cell types of the intact pancreas. The highest levels of transcription are observed in acinar cells. Transcripts of h-sgk are also detected in other cell types of the pancreas, such as pancreatic duct cells and mononuclear cells situated around the pancreatic duct. Both acinar cells and pancreatic duct cells are engaged in electrolyte transport, but acinar cells, in addition, secrete large quantities of digestive enzymes (3).
As previously shown for Hep G2 cells and Madin-Darby canine kidney
cells (17), h-sgk transcription is highly sensitive to the
volume of DAN-G cells. In Hep G2 cells, isotonic cell shrinkage by
simultaneous inhibition of Na+/H+ exchange and
Na+-K+-2Cl1 cotransport has been
shown to increase the transcription of h-sgk (17). From
this observation it was concluded that the transcription of the kinase
is sensitive to cell volume rather than osmolarity. Thus a decrease of
cell volume under the influence of secretagogues is expected to
increase h-sgk transcription. In shark rectal gland, s-sgk
transcription has indeed been observed to be enhanced by the
secretagogues vasoactive intestinal polypeptide and carbachol (16). In agreement with earlier reports (8,
13), the volume of DAN-G cells is decreased by both cAMP and
Ca2+, which are triggered by a variety of hormones. An
increase of intracellular Ca2+ in pancreatic cells yielded
the expected stimulation of h-sgk transcription, whereas cAMP
significantly decreased the mRNA levels. This latter effect cannot be
explained by cell swelling, because cAMP is shown to shrink DAN-G
cells. Thus the observations point to a cell volume-independent
inhibitory influence of protein kinase A on h-sgk transcription. The
stimulatory effect of phorbol esters and the inhibitory action of
staurosporine and calphostin C suggest protein kinase C-dependent
upregulation of h-sgk transcription. A similar stimulating effect of
PDD on h-sgk transcription has been observed previously in macrophages
(18). The effect of PDD cannot be explained by cell
shrinkage, because PDD exerts no significant effect on DAN-G cell
volume. In other tissues, stimulation by protein kinase C (4,
19) and serum (9) increase cell volume, although
the effect may vary between different tissues and cannot be
extrapolated to DAN-G cells. Nevertheless, our observations point to
cell volume-independent regulation of h-sgk transcription. Similar to
human sgk, the sgk transcription in the rat has been shown to be
stimulated by serum [20] and the abbreviation for the
kinase (representing serum glucocorticoid-regulated kinase) was
actually coined on the basis of its upregulation by serum
(20).
The marked expression of h-sgk in pancreatic epithelial cells suggests a functional role of this kinase in the regulation of epithelial transport. Coexpression of h-sgk with the epithelial Na+ channel ENaC leads to a severalfold stimulation of Na+ channel activity (2, 12, 15). As a matter of fact, h-sgk is the most powerful stimulator of ENaC hitherto described. Most likely, the kinase influences further transport systems engaged in cell volume regulation on the one side and in transepithelial transport on the other. The regulation of h-sgk by secretagogues is thus expected to modulate transepithelial transport.
In tissue specimens of patients with pancreatitis, the transcription of
h-sgk is decreased in acinar cells. Cell injury, as it occurs in
pancreatitis, is frequently paralleled by cell swelling (10), which could then account for the decline of h-sgk
transcription. Moreover, the inflammatory process could lead to
dedifferentiation of epithelial cells with subsequent loss of
cell-specific h-sgk transcription. On the other hand, pancreatitis is
characterized by a marked increase of h-sgk transcript levels in
interstitial cells within areas of pancreatic damage. It appears that
the increased abundance of h-sgk is caused by both an increased number
of h-sgk-expressing cells and increased transcript levels in those
cells. Regarding the nature of h-sgk mRNA-expressing cells, it is
likely that, because of the typical morphology, the majority of these
cells represent macrophages. Indeed, h-sgk transcription is high in macrophages and can be further stimulated in those cells by cytokines such as transforming growth factor-1 (18).
In conclusion, the present paper reveals a distinct pancreatic tissue distribution of h-sgk mRNA, with the most prominent transcription in acinar cells. Inflammatory disease of the pancreas leads to a profound redistribution of h-sgk mRNA. Northern blots reveal the expected sensitivity of h-sgk transcription to osmotic cell shrinkage and increased intracellular Ca2+ activity but reveal apparently cell volume-independent transcriptional regulation by cAMP and protein kinase C.
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ACKNOWLEDGEMENTS |
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The skilled technical support of Petra Barth, Carmen Ruoff and Sandra Bundschuh is gratefully acknowledged. The authors are indebted to Tanja Loch for secretarial help during preparation of the manuscript.
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FOOTNOTES |
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This study was supported by the Deutsche Forschungsgemeinschaft grant (No. La 315/4-3), the FORTÜNE program of the Medical Faculty of Tübingen (Nr. 302), the Federal Ministry of Education, Science, Research and Technology, and the Interdisciplinary Center for Clinical Research Tübingen (HIZKF 01 KS 9602).
Address for reprint requests and other correspondence: F. Lang, Physiologisches Institut der Universität Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany (E-mail: florian.lang{at}uni-tuebingen.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 25 February 1999; accepted in final form 23 April 2000.
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