Comparative studies of skeletal muscle proteome and transcriptome profilings between pig breeds.

Two genetically different pig breeds, the Korean native pig (KNP) and the Western meat-producing Landrace, show breed-specific traits in stress responsiveness (stress hormone levels), growth performance (live weight), and meat quality (intramuscular fat content). We analyzed expression levels within the proteome and transcriptome of the longissimus muscles of both breeds using two-dimensional electrophoresis (2-DE) and microarray analysis. We constructed a porcine proteome database focused mainly on mitochondrial proteins. In total, 101 proteins were identified, of which approximately 60% were metabolic enzymes and mitochondrial proteins. We screened several proteins and genes related to stress and metabolism in skeletal muscles using comparative analysis. In particular, three stress-related genes (heat shock protein beta-1, stress-70 protein, and heat shock 70 kDa protein) were more highly expressed in the Landrace than in the KNP breed. Six metabolism-related genes (peroxisome proliferative activated receptor alpha, short-chain acyl-CoA dehydrogenase, succinate dehydrogenase, NADH-ubiquinone oxidoreductase, glycerol-3-phosphate dehydrogenase, and sterol regulatory element binding protein-1c), all of which are involved in energy and lipid metabolism, were more highly expressed at the protein or mRNA level in the KNP breed. These data may reflect the breed dependence of traits such as stress responsiveness, growth performance, and meat quality.

Comparisons of longissimus muscle metabolic enzymes and muscle fiber types in Korean and western pig breeds.

We compared differentially expressed genes and muscle fiber types in the longissimus muscles of Korean native pigs (KNP) and the western meat-producing breeds Landrace and Yorkshire. The KNP breed exhibited a higher muscle fat content and more red meat color as determined by the a(∗) (redness) value (P<0.01) and b(∗) (yellowness) value (P<0.05) compared to the western breeds. Using differential display RT-PCR, we detected two genes that were differentially expressed in skeletal muscle among the pig breeds. These genes were identified as NADH dehydrogenase subunit 1 and ATPase subunit 6 by cloning and sequencing analysis. Both of these genes are involved oxidative phosphorylation and therefore energy metabolism. The genes were more highly expressed in the KNP breed than in the other breeds, indicating that KNPs exhibit more oxidative metabolism than do the western breeds. We also analyzed the mRNA levels of myosin heavy-chain isoforms such as type I (oxidative), type IIb (glycolytic), and types IIa and IIx (intermediate) fibers using real-time RT-PCR. The mRNA levels of oxidative and intermediate fibers were elevated in the KNP breed, whereas the glycolytic fibers were more highly expressed in the Landrace and Yorkshire pigs. These results suggest that the elevated expression of the oxidation-related metabolism genes NADH dehydrogenase and ATPase is related to meat quality as indicated by a higher content of oxidative fibers and muscle fat, as well as redder meat color.

Differential expression profiling of the proteomes and their mRNAs in porcine white and red skeletal muscles.

Skeletal muscle is an heterogeneous tissue with various biochemical and physical properties of several fiber types. In this study, we carried out the comparative study of protein expression patterns in white and red muscles using two-dimensional gel electrophoresis (2-DE). From more than 500 protein spots detected on each 2-DE gel, we screened five proteins that were differentially expressed between white and red muscles. Using peptide mass fingerprint and tandem mass spectrometry analysis these proteins were identified as myoglobin, two slow-twitch isoforms of myosin light chain and two small heat shock proteins (HSP20 and HSP27). The protein levels of myoglobin, myosin light chain and HSP20 were higher in red muscle, whereas HSP27 was higher in white muscle. In addition, genes of the identified proteins were cloned and their mRNAs were examined. Positive correlations between protein content and their mRNA levels were observed in white and red muscle. These results may provide us with valuable information to understand the different expression profiling between white and red muscle at the protein level.

Hypoxia activates signal transducers and activators of transcription 5 (STAT5) and increases its binding activity to the GAS element in mammary epithelial cells.

STATs (signal transducers and activators of transcription) are proteins with dual functions: signal transducers in the cytoplasm and transcriptional activators in the nucleus. STAT proteins act as transcription factors activated by phosphorylation on its tyrosine residues upon stimulation by various cytokines. The phosphorylated STAT molecules then form homo- or heterodimers through SH2-mediated interaction and translocate into the nucleus to activate the transcription of various target genes. STAT5 recognizes the interferon-gamma activated site TTCNNNGAA (GAS sequence) in the promoter region of the beta-casein gene. Except for prolactin-dependent beta-casein production in mammary gland cells, the biological consequences of STAT5a activation in various systems are not clear. Here we showed that STAT5a was phosphorylated 10 min after desferrioxamine (DFO) treatment, and reached a maximum induction at 4 h in mammary epithelial cells (HC11) and transfected COS-7 cells. Under hypoxic conditions (2% O2), a maximal phosphorylation of STAT5a was observed within 6 h. EMSA (electrophoretic mobility shift assay) showed that DFO or hypoxia enhanced the binding activities of STAT5a DNA to beta-casein gene promoter in mammary epithelial cells (HC11) and transfected COS-7 cells. These results showed that DFO or hypoxia induces tyrosine phosphorylation of STAT5a and also increases the binding activity of STAT5a DNA in mammary epithelial cells. Our data suggest that the STAT5 may act as a mediator in hypoxia-mediated gene expression.