Differential sorting of human parathyroid hormone after transduction of mouse and rat salivary glands

Abstract

Gene transfer to salivary glands leads to abundant secretion of transgenic protein into either saliva or the bloodstream. This indicates significant clinical potential, depending on the route of sorting. The aim of this study was to probe the sorting characteristics of human parathyroid hormone (hPTH) in two animal models for salivary gland gene transfer. PTH is a key hormone regulating calcium levels in the blood. A recombinant serotype 5 adenoviral vector carrying the hPTH cDNA was administered to the submandibular glands of mice and rats. Two days after delivery, high levels of hPTH were found in the serum of mice, leading to elevated serum calcium levels. Only low amounts of hPTH were found in the saliva. Two days after vector infusion into rats, a massive secretion of hPTH was measured in saliva, with little secretion into serum. Confocal microscopy showed hPTH in the glands, localized basolaterally in mice and apically in rats. Submandibular gland transduction was effective and the produced hPTH was biologically active in vivo. Whereas hPTH sorted toward the basolateral side in mice, in rats hPTH was secreted mainly at the apical side. These results indicate that the interaction between hPTH and the cell sorting machinery is different between mouse and rat salivary glands. Detailed studies in these two species should result in a better understanding of cellular control of transgenic secretory protein sorting in this tissue.

FIG. 1.

Secretion of hPTH from SMIE cells after transduction in vitro with Ad.hPTH. SMIE cells were incubated with the indicated MOIs of Ad.hPTH. After 48 hr, medium was harvested and assayed for hPTH by Western blot (top) and ELISA (bottom). The data shown are representative of two experiments performed in duplicate and are displayed as means ± SEM.

FIG. 2.

Distribution and biological effect of hPTH in mice after administration of Ad.hPTH to both submandibular glands by retrograde ductal instillation. Two doses were given: 1 × 10⁹ or 1 × 10¹⁰ VP/gland. Saline and an irrelevant vector, Ad.hEPO, were used as controls. Human PTH was measured by ELISA in serum (A, p = 0.004) and saliva (B) 2 days after vector infusion. The free calcium concentration in serum was determined by indirect potentiometry (C, p = 0.002) and thereafter was plotted against hPTH levels in serum to reveal a high correlation (D). Data are shown as means ± SEM (A–C) and as individual data points (D).

FIG. 3.

Production of hPTH in rats after transduction of both submandibular glands with Ad.hPTH. Ad.hPTH at either 5 × 10⁹ or 5 × 10¹⁰ VP/gland was introduced into glands by cannulating the ducts. Saline was used as a control. Two days after vector delivery, serum (A) and saliva (B, p = 0.016 for 5 × 10⁹ VP and p = 0.008 for 5 × 10¹⁰ VP, compared with control) were assayed for hPTH by ELISA.

FIG. 4.

Detection and localization of hPTH in transduced salivary glands. Two days after vector infusion, submandibular glands were removed, fixed in formaldehyde, and embedded in paraffin. Subsequent to sectioning, slides were immunofluorescently stained for hPTH (green), and phalloidin was used to visualize gland morphology (red). A transduced mouse gland is shown in (A) and a control mouse gland is shown for comparison (B). A rat gland after transduction is displayed in (C), and a control rat gland is shown for comparison (D). Arrows indicate the cellular localization of hPTH. Original magnification: 100×. Insets in (A) and (C): Enlargements of boxed areas.