Effect of transforming growth factor-a hormone-related protein on phosphate

and parathyroid transport in renal cells

LARA PIZURKI, RENE RIZZOLI, JOSEPH CAVERZASIO, AND JEAN-PHILIPPE BONJOUR Division of Clinical Pathophysiology, Department of Medicine, University Hospital of Geneva, 121 I Geneva 4, Switzerland

PIZURKI, JEAN-PHILIPPE

LARA, RENB BONJOUR.

RIZZOLI,

JOSEPH

CAVERZASIO,

AND

Effect of transforminggrowth factora) and parathyroid hormone-related protein on phosphate transport in renal ceZZs.Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F929-F935, 1990.-The decrease in plasma Pi concentration and in Pi tubular reabsorption that is often encountered in malignant hypercalcemia may be ascribed to a tumor-produced parathyroid hormone (PTH)-related protein. However, tumors are known to synthesize a variety of substances, among which is transforming growth factor-a (TGFcu). We investigated the effects of TGF-cu on Na-dependent Pi transport and on the response to PTH-related protein in cultured opossum renal epithelial cells. TGF-a! caused a concentrationand time-dependent decrease in Na-dependent Pi transport. The inhibition of Na-dependent Pi transport was detectable by 14 h of incubation and maximal by 24 h. At that time, a concentration of 10 rig/ml of TGF-cr produced a 35 t 1% inhibition. This was not associated with any change in prostaglandin production. The adenosine 3’,5’-cyclic monophosphate (CAMP) response to PTH-related protein, PTH, prostaglandin EP or forskolin, but not to pertussis toxin, was diminished in cells treated with TGF-cu for 24 h. Similar effects on Na-dependent Pi transport and CAMP production were observed in cells incubated with epidermal growth factor. The inhibition of Na-dependent Pi transport induced by either PTH-related protein or PTH was reduced after incubation with TGF-cu. Thus two different tumoral products, TGF-a and PTHrelated protein, are each capable of inhibiting Na-dependent Pi transport in cultured renal cells. Both peptides may also interact and influence the effects of each other on renal Pi transport. epidermal

growth

factor; parathyroid

hormone

homology with PTH and seemsto share the same receptor (9, 15, 17, 30). Purified material and synthetic aminoterminal fragments of PTH-related protein have been shown to mimic various effects of PTH in vivo as well as in vitro (9, 24, 29). One of these effects is to inhibit Pi transport in renal cells in a similar way to PTH, through an adenosine 3’,5’-cyclic monophosphate-dependent (CAMP) mechanism (21). This direct effect of PTHrelated protein on renal Pi transport could explain the hypophosphatemia and decreased tubular reabsorption of Pi seen in many cases of tumors associated with HM. Nevertheless, there are cases displaying a decrease in Pi reabsorption without any increase in urinary CAMP, suggesting the presence of other tumoral factors capable of influencing the renal handling of Pi. Indeed, among the different factors secreted by tumors, transforming growth factor-a (TGF-cr), its mRNA, as well as the peptide itself, has been detected in different types of tumors associated with HM and hypophosphatemia (6). TGF -a has been described as a potent bone resorbing agent, as well as an inhibitor of bone formation in vitro (11, 28, 31). The injection of TGF-a into mice has been shown to elevate plasma calcium (32). However, an influence of TGF-cu on renal Pi reabsorption and plasma Pi has not yet been investigated. In this study, we investigated the direct effect of TGF-cu, either alone or in association with PTH-related protein-( l-34), on Na-dependent Pi transport and CAMP production in a renal epithelial cell line derived from opossum kidney (OK). The OK epithelial cells possessreceptors for PTH and a Na-dependent Pi transport system, with characteristics similar to those demonstrated in brush-border vesicles isolated from kidney tubules (3). They allow assessment of the direct effects of various agents acting on Na-dependent Pi transport. Results indicate that TGF-cu alone decreased Na-dependent Pi transport. Moreover, it blunted the CAMP response to PTH-related protein and attenuated the PTH-related protein-induced inhibition of Na-dependent Pi transport.

OF MALIGNANCY (HM) may be associated with decreased tubular reabsorption of Pi and hypophosphatemia. High concentrations of extracellular calcium per se can decrease tubular reabsorption of Pi (1). However, hypophosphatemia occurring in HM may be ascribed to tumor-produced circulating factor(s) able to directly decrease the tubular reabsorption of Pi. Thus the implantation of hypercalcemic tumors in animals leads to a decreased tubular reabsorption of Pi, as shown by clearance studies (25). In vitro studies using condi- MATERIALS AND METHODS tioned medium collected from cells of a human lung squamous carcinoma have demonstrated the presence of Synthetic PTH-( l-34) (7,000 IU/mg) and isobutyla factor(s) capable of directly and specifically inhibiting methylxanthine (IBMX) were obtained from Sigma, St. Pi transport in the same manner as PTH (20). From Louis, MO. PTH-related protein-( l-34) was kindly provarious tumors, a PTH-related protein was purified and vided by Drs. B. E. Kemp and T. J. Martin, University partially sequenced, and cDNA clones were produced of Melbourne, Australia. All radioactive materials were (l&17,30). This protein displays a strong aminoterminal purchased from New England Nuclear. Dulbecco’s modHYPERCALCEMIA

036%6127/90

$1.50

Copyright

0 1990 the American

Physiological

Society

F929

Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (150.216.068.200) on January 13, 2019.

F930

TGF-cu

INHIBITS

Pi TRANSPORT

ified minimum essential medium (DMEM) and fetal calf serum (FCS) were from GIBCO, Basel, Switzerland. Rat synthetic TGF-cu was purchased from Peninsula Labs. Cell culture. The opossum kidney (OK) cells were plated at a density of 1.3 x lo4 cells/cm’ in Z-cm2 wells (CAMP assay) or 9.6-cm2 Petri dishes (Na-dependent Pi transport) and grown in DMEM containing 10% FCS, 100 IU/ml penicillin, and 100 pg/ml streptomycin. On day 6 after plating, confluent cells were incubated with TGF-a or its solvent [O.Ol N acetic acid, 5 mg/ml bovine serum albumin (BSA)] , and CAMP production and Nadependent Pi transport were determined 24 h later unless otherwise indicated. Production of CAMP. CAMP production was measured as previously described (20). Briefly, OK epithelial cells were incubated with DMEM (2% FCS) and 1 ,&i/ml [3H]adenine for 2 h. After discarding the medium, the cells were washed twice with 1 ml serum-free DMEM. The cells were incubated with 150 ,ul DMEM containing 1 mM IBMX for 10 min at 37°C. DMEM (150 ~1) with 5% heat-inactivated FCS, containing the various peptides or their solvents, were added for another 10 min. After discarding the medium, [3H]cAMP was extracted at 4°C with 5% trichloroacetic acid containing 2 mM adenosine, 2 mM ATP, 2 mM AMP, and 2 mM CAMP. After neutralization with KOH, [3H]cAMP was isolated by sequential chromatography on Dowex AG 50 W-X4 and alumina according to Salomon et al. (26). Two thousand counts/min (cpm) [ 14C]cAMP were added tq measure recovery, which was between 57 and 90%. Pi transport. The cell layers were rinsed three times with a solution at 37°C containing (in mM) 143 NaCl, 5.4 KCl, 1.8 CaC12,0.8 MgC12, and 15 N+hydroxyethylpiperazine-n’-2-ethanesulfonic acid (HEPES), pH 7.40. The cell monolayers were then incubated with 1 ml of the same solution containing 0.1 mM 32P (1 &i/ml), 0.1 g/l00 ml BSA, and 0.1 g/l00 ml D-glucose. After 4 min at 37°C the transport was stopped by rinsing the cells three times with ice-cold stop solution. The cells were then solubilized with 0.2 N NaOH, and samples were counted by scintillation. The Na-independent Pi transport in OK cells represents

Effect of transforming growth factor-alpha and parathyroid hormone-related protein on phosphate transport in renal cells.

The decrease in plasma Pi concentration and in Pi tubular reabsorption that is often encountered in malignant hypercalcemia may be ascribed to a tumor...
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