Nephrol Dial Transplant (2003) 18: III18-III22
© 2003 European Renal Association-European Dialysis and Transplant Association
Original Article
Adenovirus-mediated functional gene transfer into parathyroid cells in vivo and in vitro
1 Division of Nephrology and Clinical Research Center, Tokyo Teishin Hospital, Tokyo, 2 Division of Nephrology, Tokai University School of Medicine, Bouseidai, Isehara-city, 3 Third Department of Surgery, Tokyo Women's Medical College Hospital, Tokyo, 4 Division of Nephrology, Tokyo University School of Medicine, Tokyo and 5 Division of Nephrology and Dialysis Center, Kobe University School of Medicine, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Chronic renal failure patients usually develop secondary hyperparathyroidism and, as the disease progresses, there is a decrease in the number of vitamin D and calcium-sensing receptors (CaRs) in the parathyroid glands. Parathyroid cell function can be controlled if a functional gene is transferred into these cells using an adenovirus vector. Vitamin D or CaR genes transferred by the infected adenovirus vector induced a reduction in parathyroid hormone secretion. These results suggest that adenovirus-mediated gene transfer is a useful technique for control of parathyroid cell function.
Keywords: adenovirus; calcium-sensing receptor; functional gene transfer; vitamin D receptor
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Secondary hyperparathyroidism (2HPT) is often a complication in patients with chronic renal failure. Increased parathyroid cell proliferation results in parathyroid hyperplasia and 2HPT. Several factors promote hyperparathyroidism, such as decreased circulating concentrations of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], hyperphosphataemia, hypocalcaemia, extracellular calcium and decreased expression of the receptors for 1,25(OH)2D3 in the parathyroid gland (PTG).
Control of parathyroid hormone (PTH) gene transcription by 1,25(OH)2D3 is mediated by the vitamin D receptor (VDR). In recent studies of PTG from patients with chronic renal failure, the reduction in VDR [1] was more evident in nodular hyperplasia than in diffuse hyperplasia [2]. Calcium can also regulate PTH gene transcription. A negative regulatory element is located 3.5 kb upstream from the transcriptional factor that brings about suppression of PTH by extracellular calcium [3]. The calcium-sensing receptor (CaR) was cloned in 1993 [4], and Kifor et al. reported a decrease in CaR in the PTG of uraemic patients [5]. They observed that the lowest expression of both CaR and VDR was in the nodular portion of the gland, which is the most pathological area in HPT.
For an in vitro study of gene regulation, an established parathyroid cell line is not readily available. Dispersed bovine or human parathyroid cells have been used for in vitro studies, but they rapidly lose responsiveness to extracellular calcium ions, making it impossible to use conventional gene transfer techniques to examine gene regulation in physiological cells.
In the present study, we aimed to establish efficient techniques for rapid gene transfer into dispersed parathyroid cells and PTG of partially nephrectomized rats using an adenovirus vector, and to modulate parathyroid cell function by transferring functional genes using recombinant adenovirus vector.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Construction of recombinant adenoviruses and adenovirus-mediated gene transfer
Adenoviruses encoding the human VDR and CaR, and the Escherichia coli ß-galactosidase were constructed by the cosmid cassettes and adenovirus DNA–terminal protein complex (COS-TCP) method as described elsewhere [6]. Adenovirus harbouring the E.coli ß-galactosidase gene was designated as Ad-LacZ. Each purified recombinant adenovirus was injected into dispersed human PTG and rat PTG.
Parathyroid cell preparation and culture
Hyperplastic PTGs were surgically removed from uraemic patients with hyperparathyroidism. The glands were minced and treated with Dulbecco's modified Eagle's medium (DMEM)/F12 [5% fetal bovine serum (FBS)] containing type II collagenase (2 mg/ml) for 30 min at 37°C as described previously [7]. After filtration, dead cells and debris were removed by centrifugation on a Percoll density gradient. The cells were counted in a Burker chamber and it was observed that viability routinely exceeded 95%, as assessed by trypan blue exclusion. For culture, the cells were suspended at a concentration of 5x105 cells in 0.5 ml of DMEM/F12 (10% FBS). In each well of a 24-well dish, 5x105 cells in 0.5 ml of DMEM/F12 (10% FBS) were cultured. The cultures were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2.
Effects of adenovirus infection
On the next day, replication-deficient adenoviral vector that contained a reporter gene encoding the nuclear ß-galactosidase driven by the CAG promoter was added at 1x108 p.f.u./ml. The time course of X-gal staining was examined to analyse adenovirus infection.
Measurement of cell proliferation
In a 96-well microplate, cultured human parathyroid cells were used to determine cell proliferation with or without adenovirus infection using a commercial enzyme-linked immunosorbent assay (ELISA) system (Biotrak, Amersham Biosciences).
Measurement of PTH release
Effects of adenovirus infection on dispersed parathyroid cell function were demonstrated. PTH release was determined at 2.5 mmol/l Ca2+ by duplicate 24 h incubation of 5x105 cells in a monolayer after adenovirus infection. The concentration of PTH in the cultured medium was determined by ELISA (Immutopics, San Clemente, CA, USA).
Animals and surgical procedure
Male Sprague–Dawley rats, 7 weeks old, weighing 150–200 g were fed a vitamin D diet (Takelad) containing 0.7% Ca, 1.2% P and 0.004 U vitamin D3/g. Rats were subjected to a 5/6 nephrectomy (5/6Nx) in a two-stage procedure under ether anaesthesia. Rats were followed for 49 days and were divided into four groups depending on the type of injection into the PTG: (i) vehicle group, DMEM/F12 (FBS 10%) 5 µl (n=8); (ii) LacZ group, viral vector with Ad-LacZ 5 µl (n=8); (iii) VDR group, viral vector with Ad-VDR 5 µl (n=8); and (iv) CaR group, viral vector with Ad-CaR 5 µl (n=8).
Each solution was injected surgically under ether anaesthesia using a 31 g needle with a 10 µl syringe. Blood was collected from the jugular vein to detect serum PTH, Ca, Pi, creatinine, blood urea nitrogen and total alkaline phosphatase (ALP) concentrations both before injection and 6 days after. We also sampled all PTG detected 7 days after injection. Serum parameters, except for PTH, were measured by an auto-analyser (Hitachi Model 736-60, Hitachi Electronics Co. Ltd, Tokyo, Japan). PTH concentration was determined by commercial ELISA.
Statistical analysis
All data were expressed as the mean±SD, and the statistical analyses were performed by one-way analysis of variance (ANOVA). A P<0.05 was considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Effect of adenovirus infection in vitro
There were no differences in cultured cell number and cell proliferation with or without adenovirus infection (Figure 1a and b
). Staining with X-gal was detectable as early as 24 h after infection, and reached its peak at 48 h (data not shown). At that point, there was no difference in PTH secretion at normal calcium ion concentration with or without adenovirus infection (Figure 1c). The response of PTH secretion to calcium ion concentration was maintained. Virus solution 
1x107 p.f.u./ml was needed to infect all cells as demonstrated by X-gal staining at 48 h after infection (Figure 2).
|
|
Effect of hVDR gene transfer on PTH secretion in vitro
PTH secretion decreased significantly as the result of adenovirus infection, including the human VDR gene (Figure 3a). PTH secretion decreased depending on the 1,25(OH)2D3 concentration in the culture medium (Figure 3b).
|
Effect of gene transfer on PTH secretion in vivo
Adenovirus harbouring the E.coli ß-galactosidase gene was injected into PTG of rats with 2HPT. On day 3, adenovirus infection was detectable by X-gal staining, was maintained for 3 weeks, and the degree of infection increased with time (Figure 4). Secretion of PTH was significantly decreased by adenovirus infection, including the hCaR gene; however, other serum parameters were unchanged (Figure 5).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
There is not an established parathyroid cell line, and primary cultured cells deteriorate after a short period, so it is almost impossible to introduce a functional gene in vitro using the traditional transfection method to investigate their function. If functional genes could be overexpressed in the PTG, it would be possible to elucidate the pathogenesis of 2HPT. In the present study, we demonstrated that adenovirus-mediated target gene transfer into dispersed human parathyroid cells and rat PTG ameliorates parathyroid cell function.
Adenovirus infection of the PTG had no effect on cell number or proliferation, which suggests that adenovirus-mediated gene transfer can infect both dividing and non-dividing cells. When rapid gene transfer occurred (within 48 h), although the parathyroid cells remained responsive, a response to extracellular calcium was observed.
Infection of Ad-hVDR or Ad-CaR caused decreased PTH secretion in both cultured parathyroid cells and rat parathyroid tissue. In early renal failure, reduction in serum calcitriol and a moderate decrease in ionized calcium contribute to increased synthesis and secretion of PTH. As renal disease progresses, a reduction in parathyroid expression of VDR and CaR renders the parathyroid glands more resistant to calcitriol and calcium. Our results suggested that hVDR or CaR genes transferred by adenovirus can be translated and the target protein (i.e. VDR or CaR) increased. Further studies including confirmation using immunohistochemistry or western blot analysis are needed. It was reported recently that functional vitamin D-responsive elements have been identified in the CaR gene [10]. The suppression of PTH secretion by 1,25(OH)2D3 will be promoted by up-regulating expression of the CaR gene by 1,25(OH)2D3. However, more investigation of the action of vitamin D and altered CaR expression in 2HPT is needed; the introduction of the VDR or CaR gene using the adenovirus vector will assist in the suppression of PTH production and secretion.
Mutation or translocation of specific genes responsible for tumorous growth of the PTG have been reported in primary parathyroidism [8,9]. PRAD1/cyclin D is a well-established oncogene in which the 5'-promotor seqence of the PTH gene is translocated immediately upstream of the cyclin D gene. The stimulation of PTH gene transcription evokes the overexpression of cyclin D, leading to tumorous proliferation of parathyroid cells. In uraemia, a high dietary phosphorus also enhances PTG hyperplasia and PTH synthesis and secretion. Phosphorus restriction can prevent an increase in PTH secretion; however, the hyperplasia persists. High dietary phosphorus increases parathyroid expression of transforming factor-
(TGF-), a growth promoter [11]. Phosphorus restriction induces the cyclin-dependent kinase inhibitor p21, which arrests cell growth [12] and which is decreased in uraemic patients. It may be possible to suppress parathyroid hypertrophy or hyperplasia if the tumour suppressor gene or cell cycle regulator gene p21 is the transferring gene.
In conclusion, introduction of functional genes by adenovirus may become a useful tool in genetic regulation in parathyroid cells.
|
Notes |
|---|
Correspondence and offprint requests to: Yoshiko Iwasaki, Department of Health Sciences, Oita University of Nursing and Health Sciences, 2944-9, Megusuno, Notsuharu, Oita, 870-120, Japan. Email: ishizuka{at}oita-nhs.ac.jp, iwasakiy-tky{at}umin.ac.jp
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
- Korkor AB. Reduced binding of [3H]1,25-dihydroxyvitamin D3 in the parathyroid glands of patients with renal failure. N Engl J Med 1987; 316:1573–1577[Abstract]
- Fukuda N, Tanaka H, Tominaga Y et al. Decreased 1,25-dihydroxyvitamin D3 receptor density is associated with more severe form of parathyroid hyperplasia in chronic uremic patients. J Clin Invest 1993; 92:1436–1444
- Okazaki T, Zaijac JD, Igarashi T, Ogata E, Kronenberg HM. Negative regulatory elements in the human parathyroid hormone gene. J Biol Chem 1991; 266:21 903–910
[Abstract/Free Full Text] - Brown EM, Gamba G, Riccardi D et al. Cloning and characterization of an extracellular Ca2+ sensing receptor from bovine parathyroid. Nature 1993; 366:575–580[CrossRef][Medline]
- Kifor O, Moore FD, Wang P et al. Reduced immunostaining for the extracellular Ca sensing receptor in primary and uremic secondary hyperparathyroidism. J Clin Endocrinol Metab 1996; 81:1598–1606[Abstract]
- Miyake S, Makimura M, Kanegae Y et al. Efficient generation of recombinant adenoviruses using adenovirus DNA–terminal protein complex and a cosmid bearing the full-length virus genome. Proc Natl Acad Sci USA 1996; 7:149–158
- Rudberg C, Grimelius L, Johansson H et al. Alteration in density, morphology and parathyroid hormone release of dispersed parathyroid cells from patients with hyperparathyroidism. Acta Pathol Microbiol Immunol Scand, A 1986; 94:253–261[Medline]
- Tominaga Y, Takagi H. Molecular genetics of hyperparathyroid disease. Curr Opin Nephrol Hypertens 1996; 5:336–341[CrossRef][Medline]
- Arnold A. Genetic basis of endocrine disease 5: molecular genetics of parathyroid gland neoplasia. J Clin Endocrinol Metab 1993; 77:1108–1112[CrossRef][Web of Science][Medline]
- Canaff L, Hendy GN. Human calcium sensing receptor gene. Vitamin D responsive elements in promoters P1 and P2 confer transcriptional responsiveness to 1,25-dihydroxyvitamin D. J Biol Chem 2002; 277:30 337–30 350
[Abstract/Free Full Text] - Slatopolsky E, Brown A, Dusso A. Role of phosphorus in the pathogenesis of secondary hyperparathyroidism. Am J Kidney Dis 2001; 37:S54–S57[Web of Science][Medline]
- Dusso AS, Pavlopulos T, Naumovich L et al. p21 (WAF1) and transforming growth factor- mediate dietary phosphate regulation of parathyroid cell growth. Kidney Int 2001; 59:1182–1183[CrossRef][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||