Identification of CTLA2A, DEFB29, WFDC15B, SERPINA1F and MUP19 as Novel Tissue-Specific Secretory Factors in Mouse

Abstract

Secretory factors in animals play an important role in communication between different cells, tissues and organs. Especially, the secretory factors with specific expression in one tissue may reflect important functions and unique status of that tissue in an organism. In this study, we identified potential tissue-specific secretory factors in the fat, muscle, heart, lung, kidney and liver in the mouse by analyzing microarray data from NCBI's Gene Expression Omnibus (GEO) public repository and searching and predicting their subcellular location in GeneCards and WoLF PSORT, and then confirmed tissue-specific expression of the genes using semi-quantitative PCR reactions. With this approach, we confirmed 11 lung, 7 liver, 2 heart, 1 heart and muscle, 7 kidney and 2 adipose and liver-specific secretory factors. Among these genes, 1 lung-specific gene--CTLA2A (cytotoxic T lymphocyte-associated protein 2 alpha), 3 kidney-specific genes--SERPINA1F (serpin peptidase inhibitor, Clade A, member 1F), WFDC15B (WAP four-disulfide core domain 15B) and DEFB29 (defensin beta 29) and 1 liver-specific gene--MUP19 (major urinary protein 19) have not been reported as secretory factors. These genes were tagged with hemagglutinin at the 3'end and then transiently transfected to HEK293 cells. Through protein detection in cell lysate and media using Western blotting, we verified secretion of the 5 genes and predicted the potential pathways in which they may participate in the specific tissue through data analysis of GEO profiles. In addition, alternative splicing was detected in transcripts of CTLA2A and SERPINA1F and the corresponding proteins were found not to be secreted in cell culture media. Identification of novel secretory factors through the current study provides a new platform to explore novel secretory factors and a general direction for further study of these genes in the future.

Fig 1. Expression of adult mouse gene transcripts detected by semi-quantitative PCR.

Expressions of selected genes in fat (F), muscle (M), heart (H), lung (Lu), liver (Li) and kidney (K) were detected by PCR reaction and 1% agarose gel electrophoresis. Housekeeping gene—cyclophilin (CYC)—was used as a loading control. Genes without reported subcellular locations are marked with asterisks.

Fig 2. Different isoforms of CTLA2A and their expression in HEK293 cell culture.

A, alternative splicing of CTLA2A that leads to change in protein sequence. B, cell lysates and conditioned medium from HEK293 cells transiently transfected with HA-tagged murine expression vector of long isoform (CTLA2AL) and short isoform (CTLA2AS) and empty pcDNA3.1 vector as negative control (NC) were detected using Western blotting.

Fig 3. Different isoforms of SERPINA1F and their expression in HEK293 cell culture.

A, alternative splicing of SERPINA1F that leads to double bands in gel electrophoresis of PCR product and change in protein sequence. B, cell lysates and conditioned medium from HEK293 cells transiently transfected with HA-tagged murine expression vector of long isoform (SERPINAL) and short isoform (SERPINAS) and empty pcDNA3.1 vector as negative control (NC) were detected using Western blotting.

Fig 4. Detection of protein expression of MUP19, WFDC15B and DEFB29 in HEK293 cell culture.

A, Cell lysates and conditioned medium from HEK293 cells transiently transfected with HA-tagged murine expression vector of MUP19 and WFDC15B and empty pcDNA3.1 vector as negative control (NC) were detected using Western blotting. B, Cell lysates and conditioned medium from HEK293 cells transiently transfected with HA-tagged murine expression vector of DEFB29 and empty pcDNA3.1 vector as negative control (NC) were detected using Western blotting.

Fig 5. Expression profile in lung for CTLA2A in microarray DataSets obtained from NCBI website.

A, Expression in mice on embryonic day 12 (E12), 14 (E14), 16 (E16) and 18 (E18) and postnatal d 2 (P2), 10 (P10) and 30 (P30) in GDS3950 (n = 2 per time point). B, Expression in mice infected by E.coli and control group in GDS4583 (n = 3 per group). C, Expression in mice after 24-h infection by S.aureus deficient in alpha-hemolysin (Hla-/-) and wild type S.aureus (WT) with saline treated mice as control group (n = 3 per group). D, Expression in mice after 90-d exposure to 50 ppm arsenate (AS) with untreated mice as control in GDS4914 (n = 5 per group). E, Expression in resistant rat strain (VR) to ventilator-associated lung injury (VALI) and sensitive rat strain (VS) to VALI under treatment of high tidal volume ventilation (HV) with untreated mice as control (C) group in GDS2709 (n = 3 per group). Each bar represents mean±SEM. Statistical difference is indicated by different letters (P<0.05), * (P<0.05) and ** (P<0.01) above the bars.

Fig 6. Expression profile in liver for MUP19 in microarray DataSets obtained from NCBI website.

A, Expression in Ames Dwarf mice (n = 8) and normal mice (n = 7) with free access to standard food in GDS1261. B, Expression in 42-d old mice with growth hormone receptor knocked out (KO), truncated at amino acid residue 569 (ST) and 391 (LT) with wild type (WT) mice as control group in GDS1053 (n = 3 per group). C, Expression in C57BL/6 mouse with null mutant low-density lipoprotein receptor (LDLR) under the treatment of high fat diet for 12 wk along with WT mice as control group in GDS279 (n = 3 per group). D, Expression in mice with stearoyl-CoA desaturase 1 deficient mutants (Scd1-/-) under treatment of low-fat, high-carbohydrate diet along with WT mice as control group in GDS1517 (n = 5 per group). E, Expression in 6-wk Scd1-/- mice fed with chow diet along with WT mice as control group in GDS1374 (n = 5 per group). Each bar represents mean±SEM. Statistical difference is indicated by different letters (P<0.05), ** (P<0.01) and *** (P<0.001) above the bars.

Fig 7. Expression profile in kidney for kidney specific genes in microarray DataSets obtained from NCBI website.

A, Expression of SERPINA1F, WFDC15B and DEFB29 in mice on embryonic d 12 (E12), 13 (E13) and 16 (E16) and postnatal 10 wk in GDS2030/2031 (n = 2 per time point). B, Expression of SERPINA1F, WFDC15B and DEFB29 in postnatal C57BL/6 mice at 1, 4 and 8 wk of age in GDS4316 (n = 5 per time point). C, Expression of SERPINA1F and WFDC15B in hypertensive (HBP, n = 5), normotensive (NBP, n = 5) and hypotensive (LBP, n = 4) mice strain in GDS3675. D, Expression of WFDC15B in mice with null mutant GLIS family zinc finger 2 (GLIS2-/-) and wild type (WT) mice in GDS2817 (n = 3 per group). E, Expression of WFDC15B and DEFB29 in ureteric bud tips (UBT) and stalks (UBS) and metanephric mesenchyme (MM) from embryonic d 12 mice in GDS1583 (n = 2 per section). F, Expression of WFDC15B in mice with b-catenin overexpression (CTNNB1+) and WT mice on embryonic d 12 in GDS4449 (n = 3 per group). G, Expression of DEFB29 in homozygous mice for mutant methylmalonyl-CoA mutase (Mut-/-), heterozygous mice (Mut+/-) and WT mice in GDS4839 (n = 4 per group). H, Expression of DEFB29 in WT mice (n = 4), heterozygous mice (Cldn+/-, n = 3) and homozygous mice for mutant claudin 16 (Cldn-/-, n = 4) in GDS 3612. Each bar represents mean±SEM. Statistical difference is indicated by different letters (P<0.05) and ** (P<0.01) above the bars.