Polarized Secretion of Transgene Products from Salivary Glands in Vivo

Abstract

Previously (Kagami et al. Hum. Gene Ther. 1996;7:2177-2184) we have shown that salivary glands are able to secrete a transgene-encoded protein into serum as well as saliva. This result and other published data suggest that salivary glands may be a useful target site for vectors encoding therapeutic proteins for systemic delivery. The aim of the present study was to assess in vivo if transgene-encoded secretory proteins follow distinct, polarized sorting pathways as has been shown to occur "classically" in cell biological studies in vitro. Four first-generation, E1-, type 5 recombinant adenoviruses were used to deliver different transgenes to a rat submandibular cell line in vitro or to rat submandibular glands in vivo. Subsequently, the secretory distribution of the encoded proteins was determined. Luciferase, which has no signal peptide, served as a cell-associated, negative control and was used to correct for any nonspecific secretory protein release from cells. The three remaining transgene products tested, human tissue kallikrein (hK1), human growth hormone (hGH), and human alpha1-antitrypsin (halpha1AT), were predominantly secreted (>96%) in vitro. Most importantly, in vivo, after a parasympathomimetic secretory stimulus, both hK1 and hGH were secreted primarily in an exocrine manner into saliva. Conversely, halpha1AT was predominantly secreted into the bloodstream, i.e., in an endocrine manner. The aggregate results are consistent with the recognition of signals encoded within the transgenes that result in specific patterns of polarized protein secretion from rat submandibular gland cells in vivo.

Differential Sorting of Human Parathyroid Hormone After Transduction of Mouse and Rat Salivary Glands

“…Each sample was measured in duplicate and compared with a simultaneously performed standard curve. The serum-to-saliva distribution ratio for hPTH was calculated as described previously (Baum et al, 1999;Hoque et al, 2001).…”

“…However, salivary glands can secrete exocrine and endocrine proteins to either the apical pole or the basolateral pole of the cells (Lawrence et al, 1977;Leonora et al, 1987;Baum et al, 1999;Isenman et al, 1999). Unfortunately, the direction of this secretion is not readily predictable for all transgenic proteins.…”

Sorting of growth hormone–erythropoietin fusion proteins in rat salivary glands

“…Thus, each measured salivary transgene protein concentration, in mU per milliliter for hEpo ELISAs (or nanogram per milliliter for hGH ELISAs), was multiplied by 0.15 to yield the total amount of the protein found in saliva. For calculating total serum levels, we multiplied the determined concentration by 4 (the approximate volume of serum in mL) [17].…”

“…These lead to specific sorting routes for transgenic secretory proteins in an endocrine and exocrine manner [16]. For example, human growth hormone (hGH), which in the anterior pituitary gland is secreted into the bloodstream via the RSP, when expressed from an adenoviral vector delivered to rodent and miniature pig salivary glands, follows the tissue specific RSP, i.e., the exocrine route into saliva [17][18][19]. Conversely, transgenic human erythropoietin (hEpo) is secreted from murine salivary glands almost entirely via an endocrine route into the bloodstream [20][21][22].…”

Salivary glands and gene therapy: the mouth waters

“…On the contrary, the endocrine secretion is predominant for a1-antitrypsin. 13 A breakthrough came recently with the observation that human erythropoietin (hEPO) is secreted by ductal cells mostly if not exclusively into blood with a concentration in the saliva below the detection threshold. In an elegant study published recently in the Proceedings of the National Academy of Sciences, Voutekakis et al were able to demonstrate systemic hEPO secretion from mouse submandibular salivary glands at potentially therapeutic levels for a whole year.…”