Regulation of gap junctional communication by a pro-inflammatory cytokine in cystic fibrosis transmembrane conductance regulator-expressing but not cystic fibrosis airway cells.

Airway inflammation is orchestrated by cell-cell interactions involving soluble mediators and cell adhesion molecules. Alterations in the coordination of the multicellular process of inflammation may play a major role in the chronic lung disease state of cystic fibrosis (CF). The aim of this study was to determine whether direct cell-cell interactions via gap junctional communication is affected during the inflammatory response of the airway epithelium. We have examined the strength of intercellular communication and the activation of nuclear factor-kappaB (NF-kappaB) in normal (non-CF) and CF human airway cell lines stimulated with tumor necrosis factor-alpha (TNF-alpha). TNF-alpha induced maximal translocation of NF-kappaB into the nucleus of non-CF as well as CF airway cells within 20 minutes. In non-CF cells, TNF-alpha progressively decreased the extent of intercellular communication. In contrast, gap junctional communication between CF cells exposed to TNF-alpha remained unaltered. CF results from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Interestingly, transfer of wild-type CFTR into CF cells by adenovirus-mediated infection was associated with the recovery of TNF-alpha-induced uncoupling. These results suggest that expression of functional CFTR is necessary for regulation of gap junctional communication by TNF-alpha. Gap junction channels close during the inflammatory response, therefore limiting the intercellular diffusion of signaling molecules, and thereby the recruitment of neighboring cells. Defects in this mechanism may contribute to the excessive inflammatory response of CF airway epithelium.

Cystic fibrosis transmembrane conductance regulator does not affect neutrophil migration across cystic fibrosis airway epithelial monolayers.

Recent studies have shown that airway inflammation dominated by neutrophils, ie, polymorphonuclear cells (PMN) was observed in infants and children with cystic fibrosis (CF) even in the absence of detectable infection. To assess whether there is a CF-related anomaly of PMN migration across airway epithelial cells, we developed an in vitro model of chemotactic migration across tight and polarized CF(15) cells, a CF human nasal epithelial cell line, seeded on porous filters. To compare PMN migration across a pair of CF and control monolayers in the physiological direction, inverted CF(15) cells were infected with increasing concentrations of recombinant adenoviruses containing either the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, the DeltaF508 CFTR cDNA, or the beta-galactosidase gene. The number of PMN migrating in response to N-formyl-Met-Leu-Phe across inverted CF(15) monolayers expressing beta-galactosidase was similar to that seen across CF(15) monolayers rescued with CFTR, whatever the proportion of cells expressing the transgene. Moreover, PMN migration across monolayers expressing various amounts of mutated CFTR was not different from that observed across matched counterparts expressing normal CFTR. Finally, PMN migration in response to adherent or Pseudomonas aeruginosa was equivalent across CF and corrected monolayers. The possibility that mutated CFTR may exert indirect effects on PMN recruitment, via an abnormal production of the chemotactic cytokine interleukin-8, was also explored. Apical and basolateral production of interleukin-8 by polarized CF cells expressing mutated CFTR was not different from that observed with rescued cells, either in baseline or stimulated conditions. CF(15) cells displayed a CF phenotype that could be corrected by CFTR-containing adenoviruses, because two known CF defects, Cl(-) secretion and increased P. aeruginosa adherence, were normalized after infection with those viruses. Thus, we conclude that the presence of a mutated CFTR does not per se lead to an exaggerated inflammatory response of CF surface epithelial cells in the absence or presence of a bacterial infection.

cAMP modulates stress protein synthesis in human monocytes-macrophages.

The synthesis of heat-shock proteins (HSPs) and other stress proteins, including heme oxygenase (HO) and ferritin, is differentially induced by heat and oxidizing agents. In order to determine what role cAMP plays in those inductions in human monocytes-macrophages (m phi), we used cAMP activators or analogues alone or in combination with various stressful conditions. A stimulation in cAMP production did not per se affect stress proteins synthesis in m phi but modulated their induction in a differential way according to the stimulus. cAMP increased the synthesis of HSPs after heat shock. During erythrophagocytosis, whereas cAMP depressed the phagocytic process and the associated generation of superoxide anions, it enhanced the synthesis of HSPs, while inhibiting that of HO and ferritin. These results indicate that cAMP has a direct enhancing effect on the expression of stress proteins controlled by a classic heat-shock promoter, while decreasing their expression when induced by oxidative stress.

Actions of parathyroid hormone and parathyroid hormone-related protein.

By interacting with a structurally identical receptor, parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) display a common spectrum of action on the transport of mineral elements in bone and kidney. In vivo, PTH/PTHrP similarly reduce the renal tubular reabsorption of inorganic phosphate (Pi) and increase that of calcium. The hypercalcemic effect of PTHrP is due to an increase in both bone resorption and renal calcium reabsorption, the latter through a sodium-independent mechanism. The PTHrP-stimulated bone resorption can be totally inhibited by bisphosphonate therapy. Despite that, the fall in calcemia is moderate, indicating that the PTHrP main hypercalcemic action is due to the stimulation of the renal transport of calcium. For identical effects on renal ionic transports, PTHrP appears to less stimulate bone formation than PTH. These experimental findings are similar to clinical observations in patients with primary hyperparathyroidism or with solid malignant tumors. In vitro, the effects of PTH(1-34), PTHrP(1-34) and PTHrP(1-141) on cAMP production and sodium-dependent phosphate transport (NaPiT) are similar in kidney cells, where NaPiT is specifically inhibited by either peptide. This effect is attenuated by the competitive inhibitor [D-Trp12,Tyr34]bPTH(7-34)amide. Transforming growth factor-alpha similarly modulates the cAMP and NaPiT responses to PTH/PTHrP. In cultured mammary cells isolated from lactating rats, PTHrP elicits a 2-fold increase of cAMP production. Various products of bone and stromal cells, and of leukocytes, such as Interleukin-6 or Tumor necrosis factor-alpha, as well as high extracellular calcium concentration enhance PTHrP production by cultured lung squamous cell carcinoma and Leydig tumor cells, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation by parathyroid hormone-related protein and transforming growth factor-alpha of phosphate transport in osteoblast-like cells.

Parathyroid hormone (1-34) [PTH-(1-34)] has been shown to stimulate sodium-dependent phosphate transport (NaPiT) in UMR-106 osteoblast-like cells through a cAMP-dependent mechanism. Whether a synthetic amino-terminal fragment of parathyroid hormone-related protein (PTHrP) or the full-length molecule, which are recognized to interact with the same receptor as PTH, affect NaPiT in the same way is not known. We investigated and compared the effects of bPTH-(1-34), PTHrP-(1-34), and PTHrP-(1-141) on NaPiT and cAMP production in the osteoblastic cell line UMR-106. Each of the three peptides increased cAMP production and exerted a concentration-dependent stimulation of NaPiT after incubation for 4-6 h. We also studied the effect of transforming growth factor-alpha (TGF-alpha), which is another tumoral product secreted by certain hypercalcemia-associated tumors, on NaPiT and the TGF-alpha-induced modulation of the response to PTHrP or PTH. TGF-alpha caused a 30% stimulation of NaPiT, which remained stable from 6 to 24 h, by a cAMP-independent mechanism. In contrast, TGF-alpha attenuated cAMP production stimulated by PTH, PTHrP-(1-34), or PTHrP-(1-141). PTHrP or PTH did not further increase NaPiT in TGF-alpha-treated cells. These results indicate that NaPiT, a possibly important function of osteoblastic cells, was similarly affected by PTH and PTHrP. TGF-alpha increased NaPiT and modulated in a similar way the effects of both PTH and PTHrP.

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-produced parathyroid hormone (PTH)-related protein. However, tumors are known to synthesize a variety of substances, among which is transforming growth factor-alpha (TGF-alpha). We investigated the effects of TGF-alpha on Na-dependent Pi transport and on the response to PTH-related protein in cultured opossum renal epithelial cells. TGF-alpha caused a concentration- and 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 ng/ml of TGF-alpha produced a 35 +/- 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 E2 or forskolin, but not to pertussis toxin, was diminished in cells treated with TGF-alpha 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-alpha. Thus two different tumoral products, TGF-alpha and PTH-related 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.

Inhibition by (D-Trp12,Tyr34)bPTH(7-34)amide of PTH and PTHrP effects on Pi transport in renal cells.

We studied the effect of (D-Trp12,Tyr34)bovine parathyroid hormone-(7-34)amide [(D-Trp12,Tyr34)bPTH(7-34)NH2], a recently described highly potent antagonist of parathyroid hormone (PTH), on the adenosine 3',5'-cyclic monophosphate (cAMP) and the sodium-dependent phosphate transport (NaPiT) responses to bPTH-(1-34) or PTH-related protein [PTHrP(1-34)] in renal epithelial cells. In this system, bPTH and PTHrP increased cAMP production and inhibited NaPiT in a similar manner. (D-Trp12,Tyr34)bPTH(7-34)NH2 did not influence either cAMP or NaPiT, but markedly attenuated the responses to PTH or PTHrP. The effect was concentration dependent, and a maximal inhibition of PTH or PTHrP effects was obtained with a 10(3)- to 10(4)-fold greater concentration of the antagonist. In contrast, the same concentration of the unsubstituted counterpart, (Tyr34)bPTH(7-34)NH2, which abolished the PTH- or PTHrP-induced stimulation of cAMP production, did not affect the inhibition of NaPiT caused by either peptide. Thus (D-Trp12,Tyr34)bPTH(7-34)NH2 inhibited the effects of PTH and PTHrP on both cAMP synthesis and Pi transport in renal cells. Because of the effects of this analogue on a transport function affected by these two peptides under physiological and pathophysiological conditions, it appears to be a promising antagonist.

[The role of a parathyroid hormone analogue in the pathogenesis of malignant hypercalcemia]. / Rôle d'un analogue de l'hormone parathyroïdienne dans la pathogenèse de l'hypercalcémie maligne.

Patients with malignant hypercalcemia can display not only an increase in bone resorption, but also changes in the renal tubular reabsorption of calcium and phosphate similar to those found in primary hyperparathyroidism. A protein of tumoral origin likely responsible for this syndrome has been described. Even if produced by another gene than parathyroid hormone, it shares a homology in the aminoterminus and seems to exert a similar spectrum of action. Besides its role in malignant hypercalcemia, this analogue may be involved in physiological regulatory processes.

Action of tumoral PTH-related peptide on phosphate and calcium transport.

The recently identified parathyroid hormone-related peptide (PTHrP) exerts several actions on the transport of bone mineral elements. In vitro, the effects of the synthetic amino-terminal fragment of the parathyroid hormone-related protein PTHrP (1-34) on renal cyclic AMP production, Na-dependent Pi transport and Na+/H+ exchange appear to be very similar to PTH (1-34). In vivo, in thyroparathyroidectomized rats, PTHrP reduces the tubular Pi reabsorptive capacity (TmPi/GFR) and thereby lowers the level of plasma Pi. PTHrP (1-34) brings about a dose-dependent elevation in the level of plasma Ca. The hypercalcemic effect of PTHrP (1-34) is due to both an increase in bone Ca resorption, as assessed by an increment in fasting urinary Ca, and an elevation in renal Ca reabsorption. Like PTH, the tumoral peptide does not significantly affect the overall tubular reabsorption of Na. The PTHrP-induced increase in bone Ca resorption is completely inhibitable by diphosphonate therapy. Despite this efficacious antiosteolytic response the fall in calcemia is moderate since the marked effect of PTHrP on the renal transport of Ca is maintained under diphosphonate therapy. These experimental findings are similar to clinical observations previously made with several types of solid tumors.

Effect of synthetic tumoral PTH-related peptide on cAMP production and Na-dependent Pi transport.

Malignant hypercalcemia can be associated with a biochemical syndrome very similar to that encountered in primary hyperparathyroidism. The putative tumoral factor responsible for this syndrome has been isolated very recently from conditioned medium of a cultured lung squamous cell carcinoma (BEN), cDNA clones characterized, and an amino-terminal fragment synthesized. We investigated and compared the effect of this synthetic amino-terminal fragment of parathyroid hormone-related peptide [PTHrP-(1-34)], to purified PTHrP-(1-141) isolated from the same lung squamous cell carcinoma, and to bovine parathyroid hormone [bPTH-(1-34)] on adenosine 3',5'-cyclic monophosphate (cAMP) production and sodium-dependent phosphate transport (NaPiT) in opossum kidney (OK) epithelial cells. PTHrP-(1-34) and bPTH-(1-34) were equipotent in eliciting a 30-fold increase of cAMP production. NaPiT, as assessed by measuring the initial rate of Pi uptake, was inhibited in a concentration-dependent manner by either synthetic peptide. Half-maximal inhibition was observed with approximately 0.03-0.1 nmol/l of either bPTH-(1-34) or PTHrP-(1-34). At 10 nmol/l, either peptide produced an inhibition of 55 +/- 4 and 53 +/- 6%, respectively. This effect was specific for Pi, since the Na-dependent transport of glucose or alanine was not altered by either peptide. In OK cells dose-dependent stimulation of cAMP production and inhibition of NaPiT were also observed with purified native PTHrP-(1-141). In LLC-PK1 cells, which are devoid of PTH receptors, none of the peptides affected NaPiT. These results demonstrate a direct and specific effect of tumoral PTHrP on cAMP production and NaPiT in cultured renal epithelial cells in a way similar to bPTH.(ABSTRACT TRUNCATED AT 250 WORDS)

Factor derived from human lung carcinoma associated with hypercalcemia mimics the effects of parathyroid hormone on phosphate transport in cultured renal epithelia.

A decrease in renal tubular reabsorption of inorganic phosphate (Pi) can be observed in hypercalcemia of malignancy. In the present study we investigated the effect of serum-free conditioned medium (CM) from cells, derived from a lung carcinoma (BEN) of a hypercalcemic patient, and of PTH on cyclic AMP (cAMP) production and sodium-dependent Pi transport (NaPiT) in epithelia of two renal cell lines. In opossum kidney cells (OK), PTH is known to enhance cAMP production and inhibit NaPiT; in contrast, in LLC-PK1 cells, PTH has no effect on NaPiT since this kidney cell line is devoid of PTH receptors. In OK cells, BEN CM induced a three- to fourfold increase of cAMP production, which was blunted by the PTH inhibitors bPTH(3-34) and bPTH(7-34). NaPiT, as assessed by measuring the initial rate of Pi uptake, was inhibited in a dose-dependent manner by BEN CM, with an effect maximal between 1h30 and 6 hr of incubation (40 +/- 4% and 47 +/- 4%, respectively), corresponding to the effect produced by 1-3 nM bPTH(1-34). The Na-dependent transport of a glucose analog was affected neither by BEN CM nor by PTH. In LLC-PK1 cells, neither BEN CM nor PTH altered cAMP production nor NaPiT after 1h30 of incubation. At 6 hr, BEN CM caused a slight decrease in NaPiT. In conclusion, these results constitute the first evidence of a direct and selective inhibition by tumor-derived factor(s) of NaPiT in cultured renal epithelia. Most of the renal NaPiT inhibitory activity produced by the lung tumor required the presence of a PTH receptor-adenylate cyclase system.(ABSTRACT TRUNCATED AT 250 WORDS)