Screening of differentially expressed genes in the growth plate of broiler chickens with Tibial Dyschondroplasia by microarray analysis

Tibial dyschondroplasia (TD) is a common skeletal disorder in broiler chickens characterized by the presence of non-vascularized and unmineralized cartilage in the growth plate. Tetramethylthiuram disulfide (thiram) exerts its cytotoxic effects through membrane damage, mitochondrial injury, inhibition of glutathione metabolism, cell death, and inhibition of angiogenesis. Rath et al. [1] showed that thiram-induced TD is not produced through an increase in chondrocyte multiplication in the transition zone, nor by altering the expression of genes causing the arrest of chondrocytes in a pre-hypertrophic state. It acts by creating metabolic dysfunction that leads to the destruction of blood capillaries in the transition zone chondrocytes. Our previous studies showed that thiram could promote chondrocyte proliferation in the growth plate of chickens, and disturbs the regulation of endochondral calcification and development of normal cartilage. This results in prehypertrophic cell accumulation, angionecrosis, abnormal extracellular matrix synthesis, deferred endochondral calcification, and bone resorption (Figure 1) [2].

Previous studies have investigated the differential expression of genes related to cartilage development during late stage TD using various techniques, such as immunohistochemistry, western blotting, in situ hybridization, semi-quantitative polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), and proteomic analyses. These studies revealed abnormal expression of a variety of factors during TD, including extracellular matrix molecules (collagen, osteopontin, osteonectin), growth factors, and hormones [vascular endothelial growth factor (VEGF) and its receptor, hypoxia inducible factor-1 (HIF-1)]. Additionally, matrix metalloproteinases (MMPs), carbonic anhydrase II (CA2), and heat shock proteins (HSPs) were also shown to be affected [3–11]. Recently we generated cDNA libraries for 96-h thiram-induced broiler chicken TD using a suppression subtractive hybridization (SSH) technique, and identified 10 differentially expressed genes (DEGs). Five of these DEGs were found to be upregulated in chicken TD: matrilin-3 (matn3); chondromodulin-I (chm-1); NADH dehydrogenase (nadhdh); cytochrome C oxidase subunit III (coxIII); and enolase 1 (eno1). Differential expression of these genes may affect cartilage matrix cross-linking, angiogenesis, energy metabolism, and growth regulation [12]. The average cycle of chondrocyte proliferation to terminal differentiation is approximately 21 h; with thiram-induced cytotoxicity accompanied by rapid and significant oxidation, lipid peroxidation, and cell death. Thus, it is likely that the changes that occur during the early stages of TD initiate alterations that are found during late stage TD.

The use of DNA microarray techniques has made it possible for large-scale analysis of gene function and regulation to be conducted. DNA microarrays have been used to study skeletal development in humans and rodents [13–15]. In this study, we screened temporal changes in DEGs during early stages of TD in broiler chickens using an Affymetrix GeneChip, and verified expression patterns at different phases by qPCR. Genetic analyses were used to complement morphological changes and better understand the pathogenic mechanisms of TD.

Previous studies of TD have focused on the late stages; however, in this study we have reported the pathological changes of thiram-induced TD at earlier stages. Our findings show that the growth plate in chickens given thiram was thickened, with a decreased number of chondrocytes at the thickened proliferative and pre-hypertrophic zone. The pre-hypertrophic and hypertrophic chondrocytes exhibited pyknosis. We also observed an increased number of empty cartilage capsules and a decreased number of blood vessels, in addition to angionecrosis at the pre-hypertrophic zone. Transmission electron microscopy analysis of apoptotic cells revealed pyknotic nuclei, and dilated cisternae of the ER that were enlarged and formed a network structure; also, the cytoplasm was concentrated.

Biological process annotation revealed that the DEGs at day 1 following feeding with the thiram diet were associated with cytokine production that primarily involved the neuroactive ligand-receptor interaction pathway. The downregulated genes included chemokine (C-C motif) receptor 6 (ccr6), prostaglandin E receptor 4 (subtype EP4) (ptger4), arginine vasopressin receptor 2 (avpr2), and proprotein convertase subtilisin/kexin type 5 (pcsk5), while 5-hydroxytryptamine (serotonin) receptor 7 (htr7), and opiate receptor-like 1(oprl1) were upregulated.

Genes screened on day 2 revealed the involvement of important biological processes such as sterol metabolism, lipid biosynthesis, steroid metabolism, and terpenoid backbone biosynthesis in particular. Terpenoid, also called isoprenoid, plays an important role during cell growth. Ether lipids, also called plasmalogens or 1-O-alkyl lipids, are ubiquitous and sometimes form major parts of cell membranes in mammals. Differences between the catabolism of ether glycerophospholipids by specific phospholipases might be involved in the generation of lipid second messenger systems, which are important in signal transduction. Ether lipids can also act directly in cell signaling, as the platelet-activating factor is an ether lipid signaling molecule that is involved in leukocyte function in mammalian immune systems. Another possible function of plasmalogen ether lipids are as antioxidants to protect against oxidative stress, therefore these lipids might play a role in serum lipoprotein metabolism. This antioxidant activity comes from the enol ether double bond that is targeted by a variety of reactive oxygen species [16]. Sterols are natural active compounds that form major parts of the cell membranes and are precursors of hormones, vitamin D and sterol compound. Thus, downregulation in the expression of synthases is likely to contribute to the progression of TD.

Analysis of gene expression on day 6 revealed that the biological processes affected were associated with positive regulation of developmental processes involved in arachidonic acid metabolism and the MAPK signaling pathway. Arachidonic acid is the direct precursor of active compounds such as prostaglandin E2, prostaglandin I2 and leukotrienes C4 (LTC4). These play important roles in the regulation of lipoprotein metabolism, cell development, blood vessel elasticity and white blood cell function.

From these GO annotations at all time points, we deduced that the cytotoxic effects of thiram resulted in membrane damage, loss of enzymatic activities, and loss of protein receptor function, especially cysteine-rich proteins. This is likely to influence signal transduction, thereby leading to cell apoptosis and inhibition of pathological angiogenesis.

Our analyses also suggest that thiram-induced TD at the early stages is primarily associated with multiple metabolic pathways, focal adhesion, Jak-STAT, adipocytokine signaling pathway, GnRH signaling pathway, oxidative phosphorylation, glycolysis, Wnt, TGF-β, VEGF, and insulin signaling pathways. Metabolic pathways associated with DEGs at days 2 and 6 were identical. Impaired mitochondrial energy metabolism occurred during the initial stages of TD, with glycolysis subsequently induced at later stages of TD. The focal adhesion and JAK-STAT signaling pathways account for most DEGs, where focal adhesion and JAK-STAT may promote chondrocyte proliferation, development, anti-apoptosis, cell survival, and cell motility through Wnt or MAPK signaling pathways. During early stages of TD, many genes were found to have altered expression, due to the cytotoxic effects of thiram, therefore the proliferation of growth plate chondrocytes and metabolism were altered.

Horvat-Gordon et al. [17] studied gene expression in chondrocytes isolated from the proliferative and hypertrophic zones of the avian growth plate using microarrays. They identified a number of genes associated with chondrocyte hypertrophy. Our preliminary analyses of the seven verified DEGs revealed that lysyl oxidase (lox), which was upregulated, participates in crosslinking of extracellular collagen via oxidative deamination of lysine or hydroxylysine [18, 19]. LOX also plays important roles in cell adhesion/proliferation/migration, the intracellular signal response, and malignant processes. Moreover, LOX reenters cells and aggregates around the nuclei after oxidative changes [20]. The LOX propeptide (LOX-PP) inhibits smooth muscle cell signaling and proliferation, thereby providing a feedback mechanism to inhibit pathological angiogenesis [21]. Heat shock protein 25 (Hsp25) acts as a molecular chaperone to stabilize proteins [22–24], maintain membrane integrity, protect nucleic acids, and stabilize the cytoskeleton [25]. Hsp25 is a marker of differentiated odontoblasts [26–29] and also participates in the MAPK signaling pathway [30]. Additionally, the roles of Hsp25 in osteoblast signaling have also been widely studied [31–34]. The ID-1 protein is a member of the ID protein family that has been implicated in the regulation of cellular differentiation, cell cycle progression, senescence, and apoptosis. Moreover, ID-1 also activates VEGF by enhancing the stability and activity of hypoxia-inducible factor-1a [35, 36].

Kinectin 1 (KTN1) is a receptor protein in the reticular membrane. KTN1 mainly participates in intracellular vesicle transport and can also regulate protein synthesis in eukaryotic cells by anchoring the elongation factor-1 complex onto the ER [37]. Tamamura et al. [38] suggested that Wnt/β-catenin signaling regulates chondrocyte phenotype, maturation, and function during cartilage development. Such regulation is critical for growth plate formation, cartilage boundary definition, and endochondral ossification. Over-expression of SFRP4 may inhibit the proliferation of osteoblasts by antagonizing Wnt signaling [39]. SFRP4 may also cause CDH1 over-expression and thus affect cell adhesion and impair angiogenesis [40]. In addition, hypoxic conditions disrupt metabolism and ultimately results in ENO2 and ENO1 over-expression [12].

We identified global gene expression changes during early stages of thiram-induced TD. Downregulation of prostaglandin E receptor 4 (subtype EP4) (ptger4) and arginine vasopressin receptor 2 (avpr2) on day 1 directly influenced signal transduction and blood vessel elasticity. On day 2, downregulation of a variety of synthases altered the production of lipid compounds that are precursors of hormones, vitamin D, PTGD, and PTGE, all of which likely have an important role in angiogenesis, regulation of transcription and cell proliferation, and bone development. Taken together, the findings from this study contribute potential insights into therapies for TD, and also provide fundamental information regarding the pathological mechnisms involved during the early stages of TD.