MATERIALS AND METHODS


Isolation of the mouse Tmprss3 cDNA and gene
A mouse BAC contig that spans the region of mouse chromosome 17q syntenic to the human TMPRSS3 locus on human chromosome 21q22.3 had previously been reported (12). A 220-kb mouse BAC clone from this contig, 556K2 (CITB-CJ7-B BAC library; Research Genetics), was found to contain Tff3, which in the human genome, is approximately 60-kb centromeric to TMPRSS3. A 1-kb fragment of the mouse Tmprss3 was PCR-amplified from BAC 556K2 using degenerate primers (5'-TGYTGGAYNWSNGGNTGG-3' and 5'-CATYTGYTCRTGDATCCAATC-3' located in exon 9 amino acid position 338-343 and exon 11 amino acid position 442-448 of TMPRSS3, respectively) designed to a region of TMPRSS3, TMPRSS2 and Tmprss2 (Genbank Nos. NM_005656, AB038157, AF199362, respectively) where both the nucleotide and protein sequences were highly conserved. The resulting PCR product was cloned, sequenced and used to design Tmprss3 specific primers.
In order to isolate the full-length Tmprss3 cDNA, 5' and 3' RACE reactions were performed on poly(A)+ RNA of various mouse tissues using the Marathon cDNA Amplification Kit (Clontech). For the 3' RACE experiment, the following primers were used: (AP1 and 5'-TATCAAGTCTACAATCTATGCAGTG-3') and (AP2 and 5'-CTGAAGTGAACAAGCCTGGAGTC-3'). For the 5' RACE experiment, the following primers were used: (AP1 and 5'-TCAGGTAGCCTGCACAGAGCATG-3') and (AP2 and 5'-ACACGTCCCTGTGGTTGCAGATC-3'). A second 5' RACE experiment was performed using primers (AP1 and 5'-CACAGGCAATCTTGGCATAGTG-3') and (AP2 and 5'-ATCGGTACTCATCCTCTGCGTTC-3').
Tmprss3 introns were amplified from BAC 556K2 using primers designed within predicted Tmprss3 exons based on human genomic structure and expand long template PCR system (Roche, Mannheim). The resulting PCR products were sequenced in both orientations. Sequences were assembled using the GAP4 Staden software (13). Additional sequences were identified by BLASTN analyses of the Celera Mouse Genomic Fragments database (http://www.celera.com/) and included in the assembly.

Determination of the expression pattern of Tmprss3
RT-PCR
RT-PCR with primers 5'-CCTGGAGTCTATACGCGAATCAC-3' and 5'- ATTGGGTGGAGATCTGAGATCTG-3' located within exons 11 and 12 of Tmprss3, respectively, were performed on a cDNA panel containing 12 different cDNAs from adult mouse tissues and 4 embryonic stages to obtain a semi-quantitative estimation of the expression of the mouse Tmprss3 gene as described (14).
In order to explore the expression of Tmprss3 in the inner ear, RT-PCR was performed on RNA extracted from rat modiolus, stria vascularis, organ of Corti and cultured spiral ganglia cells, as described (15). Briefly, cochleae were dissected from 5-day-old rats, after careful removal of the organ of Corti, the modiolus was digested in Hepes buffered Eagle's media HEM, containing 0.025% trypsin and 0.001% DNase. Digested cochleae were centrifuged and the pellet was triturated in HEM containing 0.001% DNase. The auditory neurons suspension was again centrifuged and the pellet resuspended in Dulbecco modified Eagle's media (DMEM) + N1 supplement + glucose. The dissociated auditory neurons suspension was pre-plated in 35 mm tissue culture plates. The supernatant of the enriched auditory neurons was collected and cells were counted. The auditory neurons suspension was then plated in poly-DL-ornithine and laminin coated 96-well culture plates. Cell cultures were maintained at 37ºC, 10% CO2 for 3 days in serum free media.
Isolation of total RNA was performed on rat stria vascularis, modiolus and organ of Corti using Trizol (Gibco-BRL). First strand cDNA was synthesized using 1-2 mg of total RNA. Isolation of mRNA and first strand synthesis were performed on cultured rat spiral ganglia cells, untreated and treated with brain-derived neurotrophic factor (BDNF) trophic factors using the mRNA Capture kit (Roche).
PCRs for rat Tmprss3 was performed using primers designed on mouse nucleotide sequence, in areas of high homology between the human and mouse cDNAs. To detect expression of rat Tmprss3, nested PCR was performed. First PCR (220-bp product): 5'-TCCATGCTCTGTGCAGGCTAC-3' and 5'-TTCAAGTCTTCAGATCTCTCTC-3' located within exons 10 and 12, respectively. Nested PCR (163-bp product): 5'-GGCGGTGTGGACAGCTGCCAG-3' and 5'-GTGAATCCAGTCCAGGAA-3' located within exons 10 and 11, respectively.
PCRs for rat ENaC a, b and g-subunits were performed using primers designed on rat sequences (GenBank Nos. NM_031548.1, X77932.1 and U37540.1, respectively). To detect expression of the rat ENaC a- and b-subunits, nested PCR was performed. For ENaC a-subunit, first PCR (453-bp product): 5'-TCCTGCTTCCAGGAGAACAT-3' and 5'-CATCTCCACCACAGAGAGCA-3', nested PCR (406-bp product): 5'-TCAAGAAGTGTGGCTGTGCCTAC-3' and 5'-GAGCCAAACCACAGGCTCCACTG-3'. For ENaC b-subunit, first PCR (744-bp product): 5'-CAACACGACCTATTCCATCC=3' and 5'-AGCCTCAGGGAGTCATAGT-3', nested PCR (658-bp product): 5'-CCACATGATCCATAACTGCAG-3' and 5'-AATGGGCAGAGTCTGTTGGTC-3'. To detect expression of the rat ENAC g-subunit, single PCR was performed (436-bp product): 5'-GTTGCTGAATGTTCTCACCTG-3' and 5'-AGGTGGTGTGCCAGGCACCGT-3'. All first PCRs were performed in 50ml using 2ml cDNA (1/10) or captured cDNA in streptavidin tube, and 2.5U Taq Polymerase (Roche) bound to Taqstart antibody (Clontech). Nested PCRs were done using 1ml of the first PCR product as a template. A Gapdh PCR (112-bp product) using primers 5'-ACTCACTCAAGATTGTCAGCAATG-3' and 5'-GTCATGAGCCCTTCCACAATGCCA-3' was performed to illustrate the integrity of the RNA/cDNA. PCR products were separated and visualized on a 2% agarose gel.

RNA in situ hybridization
A 1.4-kb fragment from the 3'UTR of mouse Tmprss3 was amplified from mouse testis cDNA by using primers 5'-CTGAAGTGAACAAGCCTGGAGTC-3' and 5'-TAGTCATCTCTTACTGTGACTAG-3'. The resulting PCR product was cloned in TOPO TA PCRII vector (Invitrogen). Digoxigenin-labelled sense and antisense riboprobes were synthesized using T7 and SP6 RNA polymerase, respectively. Heads of 5 day-old mice were fixed for 48 h in 4% paraformaldehyde at 4°C, washed in PBS for 1 h at RT, dehydrated and paraffin embedded. Sections 8 mm thick were mounted onto superfrost slides. Slides were deparaffinated, rehydrated, acetylated (0.25% acetic anhydride, 45mM NaCl, 100mM Tris, pH 8) and washed in PBS for 5 minutes all at RT. In situ hybridization was performed overnight at 58°C in 40% formamide, 5X SSC, 1x Denhardts, 100 mg/ml tRNA and denatured labeled probes (1 mg/ml final concentration). After washing once with 2 X SSC at RT, twice with 2 X SSC and twice with 0.1 X SSC at 58°C, sections were then equilibrated with Triton washing buffer (TWB, 0.05% triton X-100, 150mM NaCl, 100mM Tris, pH 7.5), non specific binding sites were blocked with TWB containing 0.5 % blocking reagent (Roche) and sections were incubated with anti-digoxigenin antibody for 2 h at RT. After two washing steps with TWB and equilibration with alkaline phosphatase buffer NTMT (100mM NaCl, 50mM MgCl2, 100 mM Tris, pH 9.5, 0.1% tween 20) for 10 minutes, the slides were incubated with the substrate (NBT/BCIP) overnight at RT on the dark. The reaction was stopped in TE buffer and non-specific labeling was removed by incubation in ethanol 95 % for at least 1 hour at RT. Sections were dehydrated in increasing ethanol concentrations and mounted with Eukit. Pictures were taken with a Hamamatsu digital camera.

cDNA constructs
The coding region of human TMPRSS3 (GenBank No. AB038157) was PCR-amplified from thymus cDNA (Clontech) using primers, 5'-GGATGTCAGAGGTCCTGAAATAG-3' and 5'-CGGGCTGTCTTCATCACC-3' and then introduced into an EcoRV site of pBluescript II(SK+) (Stratagene) to contruct pTMPRSS3-ORF.
To visualize the subcellular localization of the TMPRSS3 protein, three different plasmids which express either tagged or wild type TMPRSS3 were constructed. Using primers 5'-ATGGGGGAAAATGATCCGCCTGC-3' and 5'-AGGTTTTTAGGTCTCTCTCCATC-3', the coding sequence of TMPRSS3 was PCR-amplified and subcloned upstream of the enhanced cyan fluorescent protein (ECFP) into vector pECFP-N1 (Clontech). Using primers 5'-CCGCCGGAATTCATGGGGGAAAATGATCCG-3' carrying a EcoR1 site and 5'-CCGCCGCTCGAGTCAGGTTTTTAGGTCTCT-3' carrying a XhoI site, the coding sequence for TMPRSS3 was PCR-amplified and subcloned into two vectors pcDNA3X(+)MycEGFP and pcDNA3X(+)HA respectively carrying a MycEGFP and a HA tag 5' to the cloning site. To make a construct for the wild type TMPRSS3 (WT-TMPRSS3), pTMPRSS3-ORF was digested with XhoI and XbaI to excise the TMPRSS3 cDNA and subcloned into pcDNA3.1 (Invitrogen).
For expression studies, pTMPRSS3-ORF was digested with XhoI and XbaI and the cDNA was subcloned into pSD5 expression vector. Single point mutants (D103G, R109W, C194F, W251C, P404L, C407R, S401A) were generated using the QuickChange site-directed mutagenesis kit from Stratagene. Sequences of the primers used are available upon request. The two truncated TMPRSS3 constructs (pro domain M1-S205: TMPRSS3 without the catalytic domain and SP domain M-C204-T454: TMPRSS3 catalytic domain only) were generated by PCR using primers 5'-CCGCCGCTCGAGGTCAGAGGTCCTGAAATAG-3' and 5'-ATAATATCTAGATCATGAGCTGTAGCCCCT-3' and 5'-CCGCCGCTGGAGGTCACCATGTGCACAGCCTGTGGTCAT-3') and 5'-CCGCCGTCTAGACGGGCTGTCTTCATCACC-3', respectively. The resulting PCR products were digested with XhoI and XbaI and subcloned into PSD5 expression vector. All the constructs described above were verified by DNA sequencing.

TMPRSS3 polyclonal antibodies.
Two polyclonal antibodies against mixture of two synthetic peptides NH2-1MGENDPPAVEAPFSFC-COOH and NH2-439CFLDWIHEQMERDLKT-COOH were raised in rabbits (CovalAb, R&B in Biotechnology; http://www.covalab.com). The immune sera were used in western blot and immunofluorescence experiments. Preimmune sera were used as negative control.

Subcellular localization of TMPRSS3 protein
Cos-7, U2OS and MDCK cells were cultured in DNEM supplemented with 10% fetal calf serum and 1% penicillin-streptomycin. Cells were grown at 37°C with 5% CO2. These cells were transfected with 0.5 mg of 5' EGFP-TMPRSS3 or 5' HA-TMPRSS3 or WT-TMPRSS3 using LipofectAMINE PLUS reagent (Life Technologies #10964-013). 5' EGFP-TMPRSS3 fluorescence was detected on 4% paraformaldehyde fixed cells using epifluorescence microscopy (Zeiss Axiophot I, color Axiocam). 5' HA-TMPRSS3 was detected on 4% paraformaldehyde fixed cells using anti-HA antibody (Santa Cruz # sc7392; dilution 1/200) followed by a rhodamine-conjugated secondary antibodies (Santa Cruz #sc-2093; dilution 1/100) and observed using epifluorescence microscopy. WT-TMPRSS3 was detected on 4% paraformaldehyde fixed cells using rabbit anti-TMPRSS3 serum (Covalab; dilution 1:400) followed by fluorescein isothiocyanate-conjugated secondary antibodies (Santa cruz #sc-2090; dilution 1:100). Images were processed with the Adobe Photoshop software.
To perform co-localisation with protein marker of known subcellular compartments, transfected cells were incubated with markers recognizing the Golgi apparatus using a mouse anti-Giantin antibody (Calbiochem #324450; dilution 1/400) followed by Rhodamine-conjugated secondary antiboby (Santa cruz #sc-2093; dilution 1/100) and the endoplasmic reticulum using a dye ER-Tracker Blue-White DPX (Molecular Probes #E-2353; dilution 1/400).
Alternatively, HeLa and Cos-7 cells were co-transfected with 1 mg of plasmid DNA of pTMPRSS3-ECFP and marker vectors including pECFP, pEYFP-Peroxi, pEYFP-Mito, pEYFP-Golgi , pEYFP-ER, and pEYFP-Mem (Clontech) using Fugene 6 (Roche Molecular Biochemicals). Transfectants were fixed in 3.5% paraformaldehyde in PBS for 30 min at 30 h after transfection, and then examined using fluorescence microscope Axioplan 2 (Carl Zeiss).

Electrophysiological Measurements in Xenopus Oocytes
Expression studies were performed in stage V/VI oocytes isolated from Xenopus laevis (Noerdhoek, South Africa). Routinely, oocytes were injected with 0.25 ng of each cRNA coding for the rat a-, b-, and g-ENaC subunits in the presence or absence of 2.5 ng of wild type or mutant TMPRSS3 cRNA in a total volume of 100nl. Oocytes were incubated in modified Barth saline (MBS) solution. 48h after cRNA injection, electrophysiological measurements were performed. Using the two-electrode voltage clamp technique, the amiloride-sensitive current (INa) was measured in the presence of 120 mM of Na+ in Frog Ringer with 5µM amiloride at a holding potential of -100 mV. The oocytes were perfused with 2 µg/ml of trypsin during 2-3 min. and INa was re-measured. All results are reported as means ±SEM. Comparing independent sets of data, unpaired t-tests were used to determine significance. In experiments where oocytes were perfused with trypsin paired t-tests were used. (n) represents the number of experiments performed.

Western blot analysis
Injected oocytes were incubated in MBS for 48h. Microsomal membrane protein extracts were obtained from pools of 10 injected oocytes lysed as described (16). Proteins were separated on a 10% SDS-polyacrylamide gel under reducing and denaturing conditions and transferred to nitrocellulose membrane. The membrane was processed using a rabbit polyclonal antibody raised against a mix of the N and C terminal ends of human TMPRSS3 according to standard procedures.