Variations of chromosomes 2 and 3 gene expression profiles among pulmonary telocytes, pneumocytes, airway cells, mesenchymal stem cells and lymphocytes

Telocytes (TCs) were identified as a distinct cellular type of the interstitial tissue and defined as cells with extremely long telopodes (Tps). Our previous data demonstrated patterns of mouse TC-specific gene profiles on chromosome 1. The present study focuses on the identification of characters and patterns of TC-specific or TC-dominated gene expression profiles in chromosome 2 and 3, the network of principle genes and potential functional association. We compared gene expression profiles of pulmonary TCs, mesenchymal stem cells, fibroblasts, alveolar type II cells, airway basal cells, proximal airway cells, CD8+T cells from bronchial lymph nodes (T-BL), and CD8+ T cells from lungs (T-LL). We identified that 26 or 80 genes of TCs in chromosome 2 and 13 or 59 genes of TCs up-or down-regulated in chromosome 3, as compared with other cells respectively. Obvious overexpression of Myl9 in chromosome 2 of TCs different from other cells, indicates that biological functions of TCs are mainly associated with tissue/organ injury and ageing, while down-expression of Pltp implies that TCs may be associated with inhibition or reduction of inflammation in the lung. Dominant overexpression of Sh3glb1, Tm4sf1 or Csf1 in chromosome 3 of TCs is mainly associated with tumour promotion in lung cancer, while most down-expression of Pde5 may be involved in the development of pulmonary fibrosis and other acute and chronic interstitial lung disease.

Telocytes differ from fibroblasts (Fbs) and mesenchymal stem cells (MSCs) as demonstrated by miR signatures and genetic profiles [15,39,40]. Proteomic signatures of the TCs are also supportive for the uniqueness and helpful in understanding of the functions. The data from omics studies demonstrated that elements within TCs are involved in (i) intercellular signalling, (ii) mechanical sensing and mechanochemical conversion task, (iii) tissue homoeostasis and remodelling/renewal, (iv) anti-oxidative stress and anti-ageing cellular mechanisms, (v) cancer cell proliferation through the inhibition of apoptosis [40,41]. Our recent work explored patterns of mouse TCspecific gene profiles on chromosome 1 and showed important roles for TCs in the prevention of tissue inflammation and fibrogenesis, development of lung inflammatory diseases or modulation of immune cell responses [42]. However, dominant patterns and specificity of gene and protein profiles of TCs which are different from other cells existed in the lung is still not completed and unclear.
The present study undertakes an in-depth analysis to find out the characters and patterns of TC-specific or TC-dominated gene expression profiles in chromosome 2 and 3, investigate the network of principle genes, and explore potential functional association. Comparisons are made among pulmonary TCs, MSCs, Fbs, alveolar type II cells (ATII), airway basal cells (ABCs), proximal airway cells (PACs), CD8 + T cells from bronchial lymph nodes (T-BL) and CD8 + T cells from lung (T-L), which may interact with TCs in the lung and trachea. Furthermore, we applied the most complete reference library of the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus database to identify key functional genes, and characteristic networks by bioinformatics tools.

Isolation and culture
Telocytes were isolated from the lung tissues of mice, primary cultured in a concentration of 1 9 10 5 cells/cm 2 , and harvested on days 5 (TC D5) and on days 10 (TC D10), as described previously [43]. RNA isolation, preparation, labelling, and hybridization were performed for DNA microarray (The Mouse 4 9 44K Gene Expression Array, Agilent, Shanghai, China). About 39,000+ mouse genes and transcripts represented with public domain annotations were gained, according to the protocol of One-Color Microarray-Based Gene Expression Analysis. The hybridized arrays were washed, fixed and scanned by the Agilent DNA Microarray Scanner (part number G2505B).
The gene expression profiles are from our earlier study, which are composed of 23,861 probes, of pulmonary TCs on days 5 and 10, Fbs and MSCs are composed of 23,861 probes [43]. There were 13,236 probes and 11,545 genes after further eliminating the probes without corresponding official symbol, which we focused on in the present study. From the total of 11,545 genes, 917 genes of the chromosome 2 and 567 genes of the chromosome 3 were analysed.

Identification of differentially expressed genes
There are about 20,000-25,000 genes in mouse, of which about 85% are similar with humans, and the propensity of functional changes was reflected in different levels of the gene expression in particular cell types. We used gene expression profiles between mouse lung cells to seek for the specific regulated and identify genes specific to TCs and their function. The fold change was utilized to identify differentially expressed genes or simply differential genes. Up-or down-regulated folds of TCs genes were calculated as compared with other cells and subtracted its own multiple of TCs, after the average of gene expression in each cell was obtained from the raw data of multi-databases, as shown in Data S1.
A set of genes are specifically up-or down-regulated in pulmonary TCs, as compared with other cells in chromosome 2 (Table 3), up-or  down-regulated genes more than 0-fold of TCs D5 were 576 or 341,  559 or 358, 228 or 689, 287 or 630, 277 or 640, 181 or 736, or 210  or 707, respectively, as compared with MSCs, Fbs, ATII, T-BL, T    In chromosome 3, 13 genes were higher than 0-fold in TCs, as compared with those in other cells (Table 4), of which 10 genes (Agl, Ecm1, Golim4, Kcnab1, Lce1a2, Nexn, Pde4dip, Plekho1, Psrc1, Rhoc, Rit1, Scamp3, Sec22b) were overexpressed 0-to 1-fold (Table 4A). Three genes Sh3glb1 (SH3-domain GRB2-like B1 -endophilin), Tm4sf1 (transmembrane 4 superfamily member 1) and Csf1 (colony stimulating factor 1) were overexpressed more than onefold, in both TC D5 and TC D10, as compared with other cells (Table 4B). 59 genes in TCs were down-regulated, as compared with other cells (Table 5). Of them, 1700013F07Rik, Amy1, Anp32e, Dnase2b, Fmo5, Pde5a, Phf17, Rwdd3 and Trim33 were down-regulated more than onefold, in both TC D5 and TC D10, as compared with other cells. In chromosome 3 (Table 6) The relationships of the more than 0-fold up-regulated genes of chromosome 2 and 3 in TC D5 and/or TC D10 were analysed by String Network analysis (www.string-db.org), as compared with other cells, to identify direct (physical) and indirect (functional) associations between selected genes of TCs. TC-specific or dominating genes in TC D5 and TC D10 were selected by up-or down-expression more than 0-fold, as compared with other cells. Figure 1A and C demonstrated the distribution of such active gene group in chromosome 2 and 3 of all cells, and interactions or potential functional links of those genes of TCs.
In chromosome 2, about 30-50% of TCs genes showed similar patterns of gene expression in MSCs, Fbs or ATII, while 5-15% of TCs genes showed similarities with ABCs, PACs, T-BL or T-L. Top 50 up-or down-regulated genes of each cell were also evaluated and their distribution within chromosome 2 genes showed the difference between them, as shown in Figure 1B. High expressed genes of each cell within chromosome 2 were evaluated and distributed as red col-  (Fig. 1B). The distribution of the high expressed genes and low expressed genes both in TC D5 and TC D10 indicates that they are in the centre of the small cluster and different from the other cells. Among the 26 co-up-expressed genes (Table 1A-C), 7 genes were found to have certain interactions (Fig. 1A).
In chromosome 3, about 50-60% of TCs genes showed similar patterns of gene expression in Fbs, MSCs, PACs or ABCs, while 0-20% of TCs genes showed similarities with ABCs, PACs, T-BL or T-L. Top 50 up-or down-regulated genes of each cell were also evaluated and their distribution within chromosome 3 genes showed the difference between them, as shown in Figure 1D. High expressed genes of each cell within chromosome 3 were evaluated and distributed as red colour (Fig. 1D). The distribution of up-expressed genes and down-expressed genes in TC D5 and TC D10 indicates that they are in the centre of the small cluster and different from the other cells. Among the 16 co-up-expressed genes (Table 4A and B), no clear or certain interactions (Fig. 1C) were found. The hierarchical cluster analysis of the differentially expressed genes (Fig. 2) clearly shows that TCs are poorly related to the other cell lines.

Discussion
Mouse chromosomes are the best studied mammalian chromosomes and are considered as gold standard of human comparative map, although genomic rearrangements occur during evolution. Certain   human disease genes were discovered by comparative genomics using the information derived from mapped mouse mutations, although they are not the simplest model for human comparison. In humans, chromosome 2 has the largest sequenced base pairs (237, 712, 649) [47], working with all of the autosomes in humans, spanning the second largest amount of total base pairs (242, 751, 149)  [48][49][50][51]. Over 31 exactly known diseases were proposed to be associated with genes on chromosome 2. In mouse, chromosome 2 is entirely sequenced and has 3146 genes encoding 1780 proteins [47], of which 917 genes were measured by bioinformatics tools in the present study. Our data demonstrated that there were 26 or 80 up-or down-regulated genes of chromosome 2 in TCs, as compared with MSCs, Fbs, ATII, T-BL, T-LL, ABCs or PACs.
One gene Myl9 (myosin, light polypeptide 9) was overexpressed most in TCs, different from other cells. Myl9 regulatory gene encodes the regulatory light chains of myosin II molecule, known to play a central role in cell adhesion, migration and division. Recent results showed Myl9 as the only gene differentially expressed in the aged versus young injured arteries [52] implying that it may be related to tissue/organ injury and ageing. Therefore, it is possible that the overexpression of Myl9 in pulmonary TCs may play an important role in lung injury and ageing. There were six genes, e.g. Pltp, Gzf1, Polr1b, Tasp1, Zbtb34 and Zfp120, down-expressed most in TCs, different from other cells. The Pltp (phospholipid transfer protein) gene is widely expressed in the body, and plays an important role in lipid metabolism, immune modulation, lipopolysaccharide binding or neurodegenerative disease [53]. Pltp is highly expressed within the lung epithelium, in chronic obstructive pulmonary disease or pulmonary inflammation [54]. TCs may play an important role of inhibiting inflammation in the lung. Roles of Gzf1 (GDNF-inducible zinc finger protein 1), Polr1b (polymerase (RNA) I polypeptide B), Tasp1

2055
(taspase threonine aspartase 1), Zbtb34 (zinc finger and BTB domain containing 34) or Zfp120 (zinc finger protein 120) genes or proteins in the lung remain unclear. Thus, there is a further need to clarify the exact mechanisms and functions of these genes in TCs.
Mouse chromosome 3 has a total number of genes of 993 which encode a total of 669 proteins [47]. Human chromosome 3 has~7% of the human genome probably related with, at least, 121 diseases that are associated with 105 genes [55] and also spanning the third largest amount of total base pairs (199,446,827) and represented about 6.5% of the total DNA in cells [56][57][58][59]. The chromosome 3 has 1550 genes, of which 567 genes of chromosome 3 were measured by bioinformatics tools in the present study. We showed that there were 13 or 59 up-or down-regulated genes of chromosome 3 in TCs, as compared with Fbs, MSCs, ATII, T-BL, T-L, ABCs or PACs. There were three genes, e.g. Sh3glb1, Tm4sf1 and Csf1, overexpressed in TCs.
Sh3glb1 gene encodes SH3-domain GRB2-like B1 or endophilin, known to have an extremely close relationship with Bax-interacting factor-1 (bif-1) [60,61], involved in cell survival and proliferation under metabolic stress and evasion of apoptosis. SH3glb1 is a membrane curvature-inducing protein interact with BECN1 though UVRAG and regulates the post-Golgi trafficking of membrane-integrated ATG9A for autophagy. At the premalignant stage, allelic loss of Sh3glb1 could enhance Myc-induced chromosomal instability and result in the up-regulation of anti-apoptotic proteins, including MCL1 and BCL2L1 [61], being responsible for enabling cells to survive and proliferate under metabolic stress and evasion of apoptosis. Endophilin is a membrane curvature-inducing protein that interacts with autophagy related beclin 1, although UV radiation resistance associated gene (Uvrag) and regulates the post-Golgi trafficking of membraneintegrated autophagy related 9A (Atg9A) protein. At the premalignant stage, allelic loss of Sh3glb1 enhances Myc-induced chromosomal instability and results in the up-regulation of anti-apoptotic proteins, including MCL1 and BCL2L1 [61]. So far, there is no reported association with any lung disease, however, we cannot exclude a role for TCs as having pro-proliferative effects through inhibition of apoptosis as showed in a previous study [41].
Tm4sf1 (transmembrane 4 superfamily member 1) is a distant member of the transmembrane 4 superfamily of cell-surface proteins characterized by the presence of four hydrophobic domains [62]. It is highly expressed in different carcinomas, e.g. in lung cancer [62], and lowly expressed in normal tissues [63]. Colony stimulating factor 1 (macrophage) (Csf1) plays an important role in cancer metastasis and invasion. It is highly expressed in different carcinomas and expressed at relatively low levels (if at all) in many normal tissues [63]. High expression of Csf1 can increase metastasis and invasion of pulmonary adenocarcinomas [64]. For example, Tm4sf1 it was up-regulated in human adenocarcinoma A549 cell line, suggesting a poor prognosis for anticancer therapy [65]. Overexpression of TM4SF1 and Csf1 in lung TCs may have a role in the development of lung cancer. Among down-expressed genes in TCs, Pde5a (cGMPspecific phosphodiesterase 5A) has an obvious association with acute and chronic interstitial lung disease. Overexpression of Pde5a may accelerate the formation of pulmonary fibrosis, while downexpression of Pde5a has important roles and effects in pulmonary fibrosis-associated pulmonary hypertension [66,67]. Therefore, we concluded that Tm4sf1 and Csf1 found to be overexpressed in lung TCs may have a role in tumour promotion. There were nine genes, e.g. 1700013F07Rik, Amy1, Anp32e, Dnase2b, Fmo5, Pde5a, Phf17, Rwdd3 and Trim33, down-expressed most in TCs, different from other cells.
Among them, only phosphodiesterase 5a (Pde5a) cGMP-specific gene is obviously associated with acute and chronic interstitial lung disease. Its high expression promotes the pulmonary fibrosis, while the inhibition of Pde5a expression ameliorates right ventricular failure and pulmonary, when is associated with bleomicin, through a reduction in reactive oxygen species [68]. Therefore, Pde5a low expression in lung TCs may have therapeutic effect on pulmonary fibrosis and other acute and chronic interstitial lung disease, probably by modulation of oxidative stress levels, as previously shown [41].
In conclusion, the present study compared genetic variations of chromosome 2 and 3 of pulmonary TCs with other related cells, e.g. Fbs, MSCs, ATII, T-BL, T-L, ABCs or PACs. Our data showed a number of TCs-specific or dominant genes in chromosomes 2 and 3, different from other lung tissue resident cells or infiltrated cells. The TCs signatures of chromosome 2 and 3 genes indicate TCs may be mainly associated with anti-inflammatory responses, the prevention of lung cancer formation and development or protective effects on pulmonary fibrosis or acute and chronic interstitial lung diseases.