Production and sorting of transgenic, modified human parathyroid hormone in vivo in rat salivary glands

Abstract

Polarized salivary epithelial cells can sort secretory proteins towards either the basolateral or apical pole. Transgenic human parathyroid hormone (hPTH) exclusively sorts apically in rat submandibular glands. To help understand this specific process we modified the hPTH cDNA sequence and delivered the cDNAs to glands in vivo using adenoviral (Ad) vectors. The Ad vectors encoded: (1) the native form of hPTH (Ad.pre-pro-hPTH1-84), (2) the native sequence, but with the pro-segment deleted (Ad.pre-hPTH1-84), and (3) a sequence containing the pre-segment followed by the first 34 amino acids of hPTH (Ad.pre-hPTH1-34). hPTH production and sorting were studied after two days. All constructs were effectively transcribed in targeted glands. However, the pre-hPTH1-84 modification led to reduced hPTH secretion and production, while no immunoreactive hPTH resulted from pre-hPTH1-34 cDNA infusion. The pre-hPTH1-84 modification had no effect on apical sorting. These in vivo results show that the signal responsible for hPTH's apical sorting does not reside in the pro-segment and that deleting both the pro-segment and the carboxyl-terminal region severely impairs post-translational processing of hPTH.

Fig.1

Schematic depiction of the different hPTH constructs encoded by and expressed from adenoviral vectors herein. (A) The full-length native pre-pro-hPTH1-84 amino acid sequence; (B) pre-hPTH1-84 with the pro sequence deleted; and (C) pre-hPTH1-34, with both the pro sequence and the sequence encoding amino acids 35-84 deleted.

Fig. 2

Transcription of three hPTH transgenes in submandibular glands in vivo. Glands were removed two days after gene transfer, homogenized and mRNA was isolated. After cDNA synthesis, reverse transcriptase (RT)-PCR was performed with primers (panel A) designed to amplify regions specific to each sequence. Panel B: RT-PCR products visualized on 1% agarose gels, comparing results from control animals (marked ‘C’ in figure) with those from rats transduced with either Ad.pre-pro-hPTH1-84 (labeled 1, 2, 3 and 4, left panel), Ad.pre-hPTH1-84 (labeled 5, 6, 7 and 8, middle panel) or Ad.pre-hPTH1-34 (labeled 9, 10, 11 and 12, right panel). As positive control (+) for each gel, the same adenoviral vector as infused in the respective animals was used directly as the PCR template. Water was used as a no template control (−). No control animal showed a positive signal, whereas all transduced animals displayed a clear and specific band of the expected size.

Fig. 2

Transcription of three hPTH transgenes in submandibular glands in vivo. Glands were removed two days after gene transfer, homogenized and mRNA was isolated. After cDNA synthesis, reverse transcriptase (RT)-PCR was performed with primers (panel A) designed to amplify regions specific to each sequence. Panel B: RT-PCR products visualized on 1% agarose gels, comparing results from control animals (marked ‘C’ in figure) with those from rats transduced with either Ad.pre-pro-hPTH1-84 (labeled 1, 2, 3 and 4, left panel), Ad.pre-hPTH1-84 (labeled 5, 6, 7 and 8, middle panel) or Ad.pre-hPTH1-34 (labeled 9, 10, 11 and 12, right panel). As positive control (+) for each gel, the same adenoviral vector as infused in the respective animals was used directly as the PCR template. Water was used as a no template control (−). No control animal showed a positive signal, whereas all transduced animals displayed a clear and specific band of the expected size.

Fig. 3

Secretion of transgenic hPTH from transduced submandibular glands. Two days after the vector infusion, serum and saliva were obtained, assayed for transgenic hPTH by ELISA, and the total secreted hPTH calculated as described in Methods. Total transgenic hPTH secreted into each compartment is displayed as mean±SEM values. Vector (5×10⁹ vp/gland) delivery to both submandibular glands led to abundant secretion of hPTH into saliva, but essentially no hPTH was detected in serum. Animals infused with Ad.pre-pro-hPTH1-84 had the highest levels of hPTH in their saliva, followed by rats that received Ad.pre-hPTH1-84. Ad.pre-hPTH1-34 gene transfer led to no detectable levels of secreted hPTH. All saline infused animals had no detectable hPTH present in their saliva. * p<0.05

Fig. 4

Detection of transgenic hPTH protein in aqueous extracts of submandibular glands. Submandibular glands were excised and homogenized in lysis buffer two days after vector delivery. Total gland protein extracted was assayed for hPTH by ELISA as described in Methods. Ad.pre-pro-hPTH1-84 mediated gene transfer led to the highest levels of hPTH measured in gland extracts. Ad.pre-hPTH1-84 delivery resulted in much lower hPTH levels, while almost no hPTH was detectable after use of the Ad.pre-hPTH1-34 vector. All values are expressed as mean±SEM values of hPTH, in pg per g extracted protein. * p<0.05