c Indian Academy of Sciences RESEARCH ARTICLE Transcriptome atlas of eight liver cell types uncovers effects of histidine catabolites on rat liver regeneration C. F. CHANG1,2,J.Y.FAN2, F. C. ZHANG1,J.MA1 and C. S. XU2,3∗ 1College of Life Science and Technology, Xinjiang University, 14# Shengli Road, Urumqi 830046, Xinjiang, People’s Republic of China 2Key Laboratory for Cell Differentiation Regulation, 3College of Life Science, Henan Normal University, 46# East of Construction Road, Xinxiang 453007, People’s Republic of China Abstract Eight liver cell types were isolated using the methods of Percoll density gradient centrifugation and immunomagnetic beads to explore effects of histidine catabolites on rat liver regeneration. Rat Genome 230 2.0 Array was used to detect the expression profiles of genes associated with metabolism of histidine and its catabolites for the above-mentioned eight liver cell types, and bioinformatic and systems biology approaches were employed to analyse the relationship between above genes and rat liver regeneration. The results showed that the urocanic acid (UA) was degraded from histidine in Kupffer cells, acts on Kupffer cells itself and dendritic cells to generate immune suppression by autocrine and paracrine modes. Hepatocytes, biliary epithelia cells, oval cells and dendritic cells can convert histidine to histamine, which can promote sinusoidal endothelial cells proliferation by GsM pathway, and promote the proliferation of hepatocytes and biliary epithelia cells by GqM pathway. [Chang C. F., Fan J. Y., Zhang F. C., Ma J. and Xu C. S. 2010 Transcriptome atlas of eight liver cell types uncovers effects of histidine catabolites on rat liver regeneration. J. Genet. 89, 425–436] Introduction histidine is either broken down into urocanic acid (UA) by histidine ammonia lyase (HAL) or decarboxylated into Liver has an unusual property of regeneration (Taub 2004). histamine (HA) by histidine decarboxylase (HDC) (Cook Partial hepatectomy (PH), a model that most clearly demon- 2001). Recent researches indicate that urocanic acid secreted strates the regenerative capacity of the liver, was first by autocrine or paracrine systems can reduce antigen present- described by Higgins and Anderson (1931). Liver regen- ing capacity of immune cells, leading to immune suppres- eration (LR) involves metabolic activities of a large num- sion (Walterscheid et al. 2006). Histamine, another degra- ber of proteins whose components of amino acids take dation product of histidine, can regulate cell proliferation part in anabolism and catabolism (Bucher 1963; Steer depending on its four receptor proteins, histamine receptor 1995). Studies have shown that histidine and its catabolites H 1, 2, 3 and 4 (HRH1, HRH2, HRH3 and HRH4) and have important physiological activities (Wu 2009), there- small G protein-coupled signalling pathways. Briefly, once fore this study is concentrated on the relevance between his- HRH1 binds to guanine nucleotide binding protein alpha q tidine metabolism and liver regeneration. Biosynthesis of polypeptide (GNAQ), glycosylphosphatidylinositol specific histidine consists of three steps: first, phosphoribosyl py- phosphorlipase C (PLC) is activated and catalyses the break- rophosphate synthetase 2 (PRPS2) catalyses ATP and down of phosphatidylinositol 4,5-bisphosphate (PIP2) into D-ribose 5-phosphate to 5-phosphoribosyl-1-pyrophosphate; inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DG). second, 5-phosphoribosyl-1-pyrophosphate receives α-ami- Further, IP3 promotes the release of Ca2+ from endoplas- no group of glutamine which is catalysed by phosphori- mic reticulum into plasma by binding it with its type-1 IP3 bosyl pyrophosphate aminotransferase (PPAT) to form his- receptor, which was anchored in the membrane of endo- tidine (Ishijima et al. 1991; Brayton et al. 1994); third, plasmic reticulum. The released Ca2+ promotes cell prolif- eration via the following signal transduction pathway Ca2+ *For correspondence. E-mail: [email protected]. RasGRF/RasGRP → Ras Raf1 → Map2k1/Map2k2 → Keywords. liver regeneration; rat genome 230 2.0 Array; histidine; histamine; urocanic acid. Journal of Genetics, Vol. 89, No. 4, December 2010 425 C. F. Chang et al. Mapk1/Mapk3 → Elk1/Myc → Srf → Fos DNA synthesis fied hepatocytes (HCs) and nonparenchymal cells-enriched (GqM pathway for short). When HRH2 binds to GNAO, supernatant fractions, respectively (Vondran et al. 2008) and. adenylate cyclase (AC) is activated to catalyse ATP to cAMP. The supernatant was mixed with equal volume of PBS, and The resulting cAMP acts as a second messenger by interact- centrifuged at 400 g for 2 × 2minat4◦C. The mixed non- ing with protein kinase and regulating other proteins to posi- parenchymal cells-rich pellet collected was adjusted to a con- tively regulate cell proliferation via ‘cAMP → PKA → Rap1 centration of 1×108 cells/mL with PBS, and mixed with 10 → Raf1 → Map2k1/Map2k2 → Mapk1/Mapk3 → Elk1/Myc μL/mL of rat anti-THY1, -GFAP, -CK31, -CD68, CD161a, → Srf → Fos → DNA synthesis’ (GsM pathway for short). -CD11c PE-antibodies, respectively. Oval cells (OCs), hep- However, HRH3 or HRH4 can block the GsM pathway and atic stellate cells (HSCs), sinusoidal endothelial cells (SECs), inhibit cell proliferation via binding to GNAO/I and repress Kupffers cells (KCs), pit cells (PCs) and dendritic cells (DCs) adenylyl cyclase activity (Demorrow et al. 2007; Huang and were picked up as previously described (Grisham 1983). On Thurmond 2008). The activities of all genes involved in the the other hand, white intrahepatic bile duct fractions left on metabolism of histidine and its derivatives are described in the nylon netting were added to the digestive solution con- detail in eight liver cell types during liver regeneration by taining 0.25% trypsin and 0.05% collagenase IV, incubated the Rat Genome 230 2.0 Array and real-time PCR (Xu and at 37◦C for 50 min, and filtered through the 200-well nylon Chang 2008), and relationships between these gene profiles netting. The filtered solution was centrifuged at 300 g for and liver regeneration were comprehensively analysed. 5 min. The resulting sediment was the pellet enriched with biliary epithelial cells (BECs) (Blair et al. 1995). The biliary Materials and methods epithelial cells were isolated with rat anti-CK19 PE-antibody 2/3 hepatectomy in rat and liver regeneration as previously described. Finally, anti-ALB and G6P, CK18 and GGT1, OC2 and OV6, CD14 and ET-1, LYZ and ED2, A total of 114 cleaning-grade Sprague-Dawley rats, weight- DES and VIM, CD8 and CD56, CD86 and CD103 antibodies ing 230 ± 20 g provided by the Animal Center of Henan Nor- were used to identify HCs, BECs, OCs, HSCs, SECs, KCs, mal University, were randomly divided into nine partial hep- PCs and DCs as previously described. atectomy (PH) groups, nine operation control groups, and one normal control (NC) group, with six rats in each group Rat Genome 230 2.0 microarray detection (male:female = 1:1). Rats in PH groups underwent opera- Total RNA was isolated from frozen livers with Trizol tion for removal of 70% of their liver as previously described reagent (Invitrogen, Carlsbad, USA) following manufac- (Higgins and Anderson 1931). Briefly, the left and median turer’s instructions and purified following the RNeasy mini lateral liver lobes were surgically removed and the animals protocol (Qiagen, Valencia, USA) (Norton 1992). The qual- were sacrificed at 0, 2, 6, 12, 24, 30, 36, 72, 120 and 168 h ity of total RNA samples was assessed by measuring the after PH, respectively. Rats in the operation control groups optical density at 260/280 nm and agarose electrophore- were treated as same as PH groups, except that their liver sis (Scott 1995). As a template, 5 μg of total RNA was lobes were all retained. The laws of Animal Protection of P. used to synthesize the first strand of cDNA using Super- R. China were strictly implemented. Script II RT (Invitrogen, Carlsbad, USA), and T7-oligo dT(24) (W. M. Keck Foundation, New Haven, USA) as Isolation and identification of eight liver cell types the primer. Second strand synthesis was performed with Rats were subjected to abdominal skin disinfection with alco- the Affymetrix cDNA single-stranded cDNA synthesis kit hol after anaesthetized by inhaling diethyl ether. The abdom- (Affymetrix, Santa Clara, USA). The cDNA product was pu- inal cavity was opened to expose the liver, and superior and rified following the cDNA purify protocol (Xiao et al. 2008). inferior vena cava was ligated followed by portal vein can- Purified cDNA, 12 μL, subsequently served as a template for nulation. Conventional two-step perfusion method was used the production of biotin labelled cRNA transcript using the to separate liver cells. Namely, the liver was perfused with GeneChip in vitro transcript labelling kit (ENZO Biochem- calcium-free perfusate preheated at 37◦C until it turned grey, ical, New York, USA). The labelled cRNA was purified us- then with a 15 mL 0.05% collagenase IV solution instead of ing the RNeasy Mini Kit columns (Qiagen, Valencia, USA) perfusate at a flow rate of 1 mL/min. After liver capsule was (Kube et al. 2007). The concentration, purity and quality of removed, the perfused liver was cut into small pieces and di- cDNA and cRNA were assessed as above. cRNA (1 μg/μL), gested with 0.05% collagenase IV for 15 min at 37◦C. After 15 μL was incubated with 6 μL5× fragmentation buffers and filtering through the 200-well nylon netting, the liquid was 9 μL RNase free water for 35 min at 94◦C, and digested to centrifuged at 500 g for 3 min. Pellet at the bottom was col- 35–200-bp cRNA fragments. The prehybridized Rat Genome lected and washed three times in a 4◦CPBSbuffer to adjust 230 2.0 microarray was placed into a hybridization buffer the cell concentration to 1×108 cells/mL. Mixed cell suspen- prepared following the Affymetrix protocol, and hybridized sion (6 mL) was spread onto the surface of 4 mL 60% Percoll in a rotating chamber (60 rpm; 16 h; 45◦C).