ECR-MAPK Regulation in Liver Early Development

Early growth is connected to a key link between embryonic development and aging. In this paper, liver gene expression profiles were assayed at postnatal day 22 and week 16 of age. Meanwhile another independent animal experiment and cell culture were carried out for validation. Significance analysis of microarrays, qPCR verification, drug induction/inhibition assays, and metabonomics indicated that alpha-2u globulin (extracellular region)-socs2 (-SH2-containing signals/receptor tyrosine kinases)-ppp2r2a/pik3c3 (MAPK signaling)-hsd3b5/cav2 (metabolism/organization) plays a vital role in early development. Taken together, early development of male rats is ECR and MAPK-mediated coordination of cancer-like growth and negative regulations. Our data represent the first comprehensive description of early individual development, which could be a valuable basis for understanding the functioning of the gene interaction network of infant development.


Introduction
Development is differential expression of the genome of organisms in different time points. Mammalian liver especially plays a vital role in the coordination of various physiological processes, and due to the different metabolic needs for male and female reproduction, mammalian liver shows considerable sexual dimorphism; this phenotypic expression is mediated via sex hormones [1]. Androgen response of the rat liver shows notable change during individual development and only the postpubertal adult (40-750 days of age) is subject to androgen-inducible genes and androgen-repressible genes [2]. Thus, transcriptional control in mammals must be properly coregulated for early stages of liver formation, perinatal repression, and position-dependent regulation [3]. Furthermore, expression profiles of fetal and natal liver tissues from mice reveal two stages during embryonic liver development; embryonic day 14.5 is a transition point when hepatocytes occur. Postnatal processes are also divided into two stages (ΙΙΙ and ΙV) and genes expression profile of stage ΙV (ranging from day 7 to week 18) exhibited more invariant property [4].
Previous researches focus on embryonic development, using targeted methods such as genetic modification, quantitative PCR, hybridization, and electrophoresis. However, early growth is less concerned. Early growth consists of natal, prepuberty, puberty, and adult stages. And moreover, early growth is connected to a key link between embryonic development and aging [4]. Therefore it is necessary to deepen the study of the early development of individual growth and thus to provide a basis and reference for dietary intake and disease prevention and control in the process of human growth, especially infant.
Utilizing systems biology approaches, for example, by combining global gene expression profiling and metabolic pattern techniques, provides means to determine characteristic transcript profile and endpoint metabolic effects of development. Integrated information from transcriptomic and metabonomic profiling contributes to elucidate mechanisms of a developmental effect in detail and with comprehensiveness.
This research compared the gene expression profiles of 22 days (3 weeks) and 16 weeks of age, using Wistar rat as a model from public database, and furthermore clinical biochemistry, qPCR, cell culture, and NMR were carried out for validation and confirmation from independent animal experiment, to reveal temporal migration information and the transcription pattern of this early growth process.

Animal Experiment and Cell
Culture. This study partially came from public database GSE32156 [5]. Briefly, offspring from Wistar Han dams were fed normally. Liver samples were collected at postnatal day (PND) 22 ( = 5) and week 16 ( = 5) of age for liver gene expression profile analysis. Independent animal experiment was carried out according to guidelines of the government of China. Sera for clinical biochemistry, urines for NMR, and livers for qPCR and cell culture were collected when the rats were decapitated after anesthesia with isoflurane. Rat primary liver cells were cultured and dexamethasone (dex, 0.1 M) or cycloheximide (CHX, 0.05 g/mL) was added as indicated.

Transcriptomic Analysis.
Total RNA was isolated from 10 rat livers, 5 from the control pups and 5 from the adults, with Trizol Reagent (Invitrogen Corp., Carlsbad, CA), in accordance with the manufacturer's instructions. The concentration and purity of total RNA were determined by spectrophotometer, 28S and 18S rRNA. The Affymetrix Rat Genome 230 2.0 arrays were used to monitor variations in gene expression profile. The log-transformed (base = 2) data were obtained for all probes and array-wise normalized using Affymetrix Dchip 2006.
The t-test and Wilcoxon signed-rank test were utilized for significance analysis of microarrays (SAM) [6][7][8][9]. A permutation test was employed for estimating the falsediscovery rate (FDR < 0.05, = 200 ∼500). The CapitalBio Molecule Annotation System (MAS), KEGG, and GenMAPP databases were used for pathway analysis (http://bioinfo.capitalbio.com/mas). For each pathway, genes with known rat orthologues were compared with sets of significant genes from SAM to define the effects of corresponding pathway.
The relationship of genes or gene clusters was carried out using Pearson's correlation, Spearman's correlation, or 2D STOCSY (statistical total correlation spectroscopy).

Quantitative Real-Time PCR and Western
Blot. cDNA was synthesized using an oligo-(dT)15 primer (Invitrogen). PCR primers were designed with Primer Premier 5.0 software. The housekeeping gene -actin was used as an internal control. The PCR amplification was conducted at 95 ∘ C for 15 min, followed by 40 cycles of 94 ∘ C for 5 s, 58 ∘ C for 15 s, and 72 ∘ C for 10 s. The relative mRNA levels of selected genes were calculated with the 2 −ΔΔCt method [10]. Liver proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to blotting membrane. Immunoblots were blocked with 3% bovine serum albumin in Tris-buffered saline/Tween-20 buffer for 60 min at room temperature and incubated overnight at 4 ∘ C with primary antibodies. Blots were developed by an Enhanced Chemiluminescence Western blotting kit (Amersham Biosciences, Uppsala, Sweden) and visualized by a Gene Genome bioimaging system. Bands were analyzed by densitometry with GeneTools software (Syngene, Frederick, MD, USA). Values were reported as means ± SD. Statistical differences were determined by the one-way ANOVA multiple range test and the Wilcoxon rank sum test. Statistical significance was set at < 0.05.

NMR Spectroscopy
Acquirement. 550 L urine was mixed with 55 L of phosphate buffer, followed by centrifugation. 1D 1 H NMR spectra were acquired (298 K, Bruker Avance III-600 MHz NMR spectrometer) with 32 transients for urine using a standard presaturation pulse sequence (presaturation during a relaxation delay and during the mixing time). 2D J-resolved, 1 H-1 H correlation, total 1 H-1 H correlation, 1 H-13 C heteronuclear single quantum coherence, and 1 H-13 C heteronuclear multiple bonds correlation NMR spectra [11][12][13] were acquired for selected urine to assign metabolites.
2.6. Statistical Analysis of NMR Spectra. NMR spectra were processed routinely [14] for phase, baseline, and chemical shift reference calibrations.
Unsupervised PCA (principal component analysis) was performed (SIMCA-P 11.0 demo, Umetrics, Sweden) to outline intrinsic similarity/dissimilarity within the data set scaled to unit variance (UV). Comparisons between temporal animals were made by carrying out qualitative PLS (partial least square regression) and O-PLS (orthogonal projection to latent structures) models (class information as qualitative Y variable). The validity of the models was assessed by 2 (predictability) and 2 (interpretability) of the model. Meanwhile, the same models were validated by a 7-fold cross validation [15], cross validation-ANOVA [16][17][18], and a permutation test ( = 200) [19]. Valid models including significantly changed metabolites (denoted by red color) were visualized and shown in the colored correlation coefficient loading plots (MATLAB version 7.1, Mathworks Inc; Natwick, USA).

Weight and Clinical Biochemistry of Early Individual
Development. Adult rats (∼313.6 g) have much more weight than pups (∼219.3 g) ( < 0.001, Table 1). Serum clinical biochemistry data from the adult rats contained higher levels of metabolites, such as glucose, triglyceride, testosterone, and lower enzymes, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (AlkP) compared with those from the pup rats ( < 0.05, Table 1).
3.6. qPCR Validation of Early Individual Development. qPCR validation for highlighted microarray genes was carried out for pups and adults. The results demonstrated that the mRNA level of obp3, a major regulator in odorant binding, was elevated 3.75-fold in the adult group compared to pups; the expression levels of rup2, hsd3b5, dhrs7, cyp2c13, ust5r, stac3,  zfp37, ppp2r2a, socs2, atp6ap2, pik3c3, and ghr were elevated significantly in the adult rats compared to the pups, while the expression levels of pcdh17, abcg8, ccna2, s100 g, cxcl13, tox, and akr1b7 were decreased significantly (Figure 2(a), < 0.01).

Obps and Its Related Proteins.
In order to characterize opb3 protein and its related proteins, we assayed Western blotting. The expression levels of obp3, hsd3b5, ppp2r2a, socs2, and pik3c3 proteins were elevated significantly in the adult rats compared to the pups, while the expression levels of cxcl13, tox, and akr1b7 proteins were decreased significantly (Figure 2(d), < 0.01).

Metabolic Profile of Temporal Rats.
Using PLS, invalid models indicated that adults were metabolically stationary from week 15 to week 19 of age (Table 4). With age (from week 8 to week 13 of age), taurine and octanoate (8 : 0) were increased, whilst succinate was lowered ( < 0.05, Figure 3).

Discussions
Early development is a physiology process, and we found that in this early individual development, extracellular region and space (ECR)-SH 2 containing protein-MAPK pathway plays a vital role. Meanwhile, early individual development is ECR and androgen-mediated feedforward coordination network of positive cancer-like growth and negative regulations.

Androgen-Responsive Genes.
Androgen-dependent 2u globulin (obp3) is a group of low molecular weight (Mr ∼18,000) male specific urinary proteins synthesized and secreted by hepatocytes. In the male rat, hepatic synthesis of 2u globulin begins at puberty (∼40 days), reaches a peak level (∼20 mg/day) at about 75 days, and declines during old age [21]. Age-dependent changes in the expression of androgen-responsive genes (alpha 2u globulin) reflect changing androgen sensitivity [2].   Accordingly, androgen receptor at week 16 was upregulated 1.25 times than at week 3 ( = 2.4 − 05).

Female-Prefer Genes.
Female-specific tox changes in gene expression during postnatal liver development reflect the deceleration of liver growth and the induction of specialized liver functions, with widespread changes in sex-specific gene expression primarily occurring in male liver [25].
Male and female genes are both increased, but their magnitudes in male are larger than that in female-change fold of male gene obp3 is 3.75 times more at senior than at junior.
Overlap between embryonic liver development and liver cancer is not only in cell cycle or apoptosis, but also in metabolic pathways associated with carbohydrate and lipid metabolism [30]. Fetal hepatocytes have high IGF2 and E2F3 expressions, and levels of IGF2 and E2F3 mRNA were positively correlated to human prostate and bladder cancers [31]. However, fetal and infant livers have no cancers. [23], cdkn1a [32], rgs3 [22], cish, spink3, cyp17a1, and nfe2 [33] were involved in negative Redox/organization Transcription MAPK Immune Extracellular Membrane-SH 2 /RTK/GPCR Figure 4: ECR-MAPK-mediated early individual development network. Extracellular region and space (ECR) act as nutrition ligand and information input. Ligands interact with membrane transports and SH 2 -containing/MAPK related signals and regulate cell cycle, transcription, and proteolysis, leading to short-term steroid, fatty acid biosynthesis, redox and metabolic process, and long-term collagen development and organization. G protein coupled receptors/G protein, catalytic receptors, and ECR signals converge at MAPK cascades.

Negative Control Genes. Socs2
control. Cancer-like early individual development, but no cancer, is maybe due to counteracting effects of negative control and cooperation of the two sides.

Feedforward Regulation.
Feedforward regulation in pheromone-activated MAPK pathway ensures stability and rapid reversibility of a cellular state [34].
Cxcl13, belonging to extracellular region [35], takes part in positive regulation of cytosolic calcium ion concentration and immune response [36].
Nonzero uterus dependent initial conditions allow fast early development and sensing, and meanwhile, feedforward modulations appear at reversible developmental transitions, because this network control can obtain the aims of growth stability and rapid reversibility without loss of external signaling information [34].
In a summary, qPCR validation was for gene expression profile, and meanwhile, cell induction/inhibition assays, Western blot, and NMR-based metabonomics were carried out for confirmation of gene results. Using dynamic assays of body weight, serum biochemistry, transcript, protein, and metabolite profile, we reveal that, in early individual development, increasing magnitude in male is larger than that in female, and cancer-like growth coordinates negative regulation; meanwhile, feedforward modulations appear at developmental transitions, obtaining aims of growth stability and rapid reversibility without superoxidation or maglinant growth; more importantly, extracellular matrix-kinase cascade responses play a vital role in this early individual development. Taken together, extracellular matrix-kinase cascadebased feedforward cooperation of cancer-like growth and negative regulation realize win-win long-term growth stability and short-term rapid reversibility/fluctuation in gradual transition of early individual development. This finding is particularly important for understanding the gene expression network of infant development.