Salivary Gland Secretion Mechanisms During Normal And Altered Functional States

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Saliva maintains oral health. Building on our past studies of saliva formation and its alteration during pathology, we previously developed novel approaches to treat salivary dysfunction using principles of gene therapy, as well as strategies to use normal salivary glands as a gene transfer target site for treating systemic single protein deficiency disorders (SSPDDs). Our studies during the past year primarily addressed fundamental questions necessary for salivary gland gene therapy to move successfully into the clinic for phase-I trials. Treatment of most head and neck cancer patients includes irradiation (IR). Salivary glands in the IR field suffer irreversible damage. We have focused much of our effort on trying to restore function to damaged glands, as well as to prevent such damage during IR. Based on previous pre-clinical studies with a serotype 5 adenoviral (Ad5) vector encoding human aquaporin-1 (AdhAQP1) conducted in rats and miniature pigs, and an extensive safety study of AdhAQP1 in rats, as previously reported, we submitted a clinical protocol for testing AdhAQP1 in patients that received all required approvals, including that of the FDA. After establishing the infrastructure for conducting this protocol, during the past year we began screening patients and determining enrollment eligibility according to approved inclusion and exclusion criteria. Recently, we delivered the AdhAQP1 vector to our first patient. In collaboration with the Radiation Biology Branch of NCI, we continue to examine the usefulness of non-gene therapy and gene therapy approaches to prevent IR-induced oral tissue damage in mouse models. For example, using the stable nitroxide Tempol, we previously reported considerable success in providing salivary gland radioprotection without tumor protection. Of note this year, in related studies, we began to evaluate Tempol utility for protecting oral mucosa from IR-induced mucositis, using both single dose and fractionated IR schemes. Tempol effectively prevented IR-induced oral mucositis with both IR schemes. Also, initial studies performed this year suggest that a gene therapy approach, using the keratinocyte growth factor cDNA delivered to salivary glands, can substantially reduce IR-induced oral mucositis. Our past studies in mice clearly showed that salivary glands are a potentially valuable gene transfer target site for treating SSPDDs. However, the conduct of large animal studies is a critical step in the development of a clinical gene therapy. As we reported last year, the serum: saliva distribution, i.e., sorting, of transgenic human erythropoietin (hEpo) differs markedly between mice (primarily endocrine), miniature pigs (primarily exocrine), rats and rhesus macaques (similar to each other and intermediate to mice and miniature pigs). Thus, at least for the model constitutive pathway secretory protein hEpo, there are significant species-specific differences in sorting that must be understood prior to any clinical application. During this past year we have conducted similar protein sorting studies using an Ad5 vector encoding human growth hormone (AdhGH). We have long-used hGH as a regulated secretory pathway (RSP) model transgenic secretory protein. After AdhGH salivary gland administration, almost all hGH was secreted into saliva in mice, rats, rhesus macaques and minipigs. Thus, although differences in the sorting of hEpo exist between the four animal models studied, for all species hGH was rigorously sorted via an exocrine pathway into saliva. Interestingly, the constitutive pathway is classically described as a default secretion pathway (i.e., random). The mechanisms responsible for sorting secretory proteins (versus membrane proteins) are not clear for any cell type, and it seems reasonable to hypothesize that our in vivo results reflect important differences in the expression and/or metabolic fate of key molecules essential to the recognition and sorting of the hEpo protein within salivary glands of mice, rats, minipigs and macaques. Based on our studies, we hypothesize that mouse and rat submandibular glands, which are in general structurally and physiologically similar, have significant inherent differences in one or more key intracellular molecule(s) utilized in sorting hEpo for secretion. We further hypothesize that identification of these differences will lead to useful strategies to employ for enhancing clinical applications of salivary gland gene transfer for SSPDDs. Also, this year, we began to investigate the sorting characteristics of other model secretory proteins after their expression as transgenes in mouse and rat salivary glands. The first molecule chosen was human parathyroid hormone (hPTH), a critical hormone for systemic calcium homeostasis, which is normally secreted via the RSP into the bloodstream from the parathyroid glands. An Ad5 vector containing the hPTH cDNA was constructed and administered to submandibular glands of both mice and rats and, thereafter, the secretion of hPTH measured in saliva and serum. Two days after vector delivery, high levels of hPTH were found in serum of mice, leading to elevated serum calcium levels. Only low amounts of hPTH were found in saliva, quite unlike the results observed with hGH. Conversely, two days after vector infusion in rats, an extensive hPTH secretion was detected primarily in saliva, with little secretion into serum, i.e., exactly like hGH. Confocal microscopy showed hPTH in the transduced glands localized basolaterally in mice and apically in rats, consistent with the hPTH serum: saliva distributions in these species. As with hEpo reported last year, it appears that the interaction between hPTH and the cell sorting machinery is different in mouse and rat salivary glands. We anticipate that detailed studies of hPTH secretory protein sorting will also facilitate our understanding of this process in salivary glands. When we began these studies many years ago, one of our original hypotheses was that because of the absence of key amino acid sequence signals, which are necessary for RSP sorting, hEpo secretion would be directed predominantly into the bloodstream in all species. Although this hypothesis was supported by then existing dogma, based on a large body of in vitro results with cultured endocrine cell lines, as noted above our in vivo results showed hEpo sorting from salivary glands to be more complex. However, during the past year we demonstrated that amino acid sequence signals obtained from hGH can considerably influence hEpo sorting. Specifically, this was shown experimentally through studies examining the sorting of hEpo and hGH fusion proteins in mice and rats. The most interesting findings came from the rat studies. We constructed and expressed two fusion proteins, hEpo-hGH and hGH-hEpo, using Ad5 vectors, and delivered them to rat submandibular glands in vivo via retroductal cannulation. 72 h following vector administration serum and saliva were collected and assayed for hEpo levels. The ratios of the total amount of immunoreactive hEpo in saliva to that found in saliva plus serum were then calculated. Higher ratios then indicate secretion of the construct preferentially into saliva, i.e., via an exocrine route. While the ratio from animals administered AdhEpo alone had a mean value of 0.396 (median 0.343), those of rats treated with either AdhEpo-hGH or AdhGH-hEpo had mean values of 0.886 and 0.808, respectively (median 1 and 1). The differences in these values between the control vector (AdhEpo) and the two experimental groups were highly statistically significant. Thus, these in vivo studies demonstrate for the first time that hGH sequence, in an N- as well as C-terminal position, can influence the secretion of a constitutive pathway protein.