Cellular localization of group IIA phospholipase A2 in rats.

It has been known that group II phospholipase A2 (PLA2) mRNA and protein are present in the homogenates of the spleen, lung, liver, and kidney in normal rats, but the cellular origin of this enzyme has not been yet identified. At present, five subtypes of group II PLA2 have been identified in mammals. Antibodies or mRNA probes previously used for detecting group II PLA2 need to be evaluated to identify the subtypes of group II PLA2. In this study we tried to identify group IIA PLA2-producing cells in normal rat tissues by in situ hybridization (ISH) using an almost full-length RNA probe for rat group IIA enzyme. Group IIA PLA2 mRNA was detected in megakaryocytes in the spleen and Paneth cells in the intestine by ISH. These cells were also immunopositive for an antibody raised against group IIA PLA(2) isolated from rat platelets. Group IIA PLA2 mRNA-positive cells were not detected in lung, liver, kidney, and pancreas. Under normal conditions, group IIA PLA2-producing cells are splenic megakaryocytes and intestinal Paneth cells in rats.     

Enhanced expression of TGF-beta and c-fos mRNAs in the growth plates of developing human long bones

The expression of mRNAs for type I and type II procollagens, transforming growth factor-beta (TGF-beta) and c-fos was studied in developing human long bones by Northern blotting and in situ hybridization. The cells producing bone and cartilage matrix were identified by hybridizations using cDNA probes for types I and II collagen, respectively. Northern blotting revealed that the highest levels of TGF-beta mRNA were associated with the growth plates. By in situ hybridization, this mRNA was localized predominantly in the osteoblasts and osteoclasts of the developing bone, in periosteal fibroblasts and in individual bone marrow cells. These findings are consistent with the view that TGF-beta may have a role in stimulation of type I collagen production and bone formation. Only a low level of TGF-beta mRNA was detected in cartilage where type II collagen mRNA is abundant. In Northern hybridization, the highest levels of c-fos mRNA were detected in epiphyseal cartilage. In situ hybridization revealed two cell types with high levels of c-fos expression: the chondrocytes bordering the joint space and the osteoclasts of developing bone. These differential expression patterns suggest specific roles for TGF-beta and c-fos in osseochondral development.     

Expression of nuclear genes encoding the urea cycle enzymes, carbamoyl-phosphate synthetase I and ornithine carbamoyl transferase, in rat liver and intestinal mucosa

RNA dot-blot, quantitative electron microscope immunocytochemistry, and electrophoretic immunoblotting techniques were employed to investigate the expression of carbamoyl-phosphate synthetase I (CPS) and ornithine carbamoyl transferase (OCT) genes in rat liver and intestinal mucosa. Comparing only those cell types in the two tissues which express these enzymes, we show that the concentration of CPS and OCT in hepatocyte mitochondria is 2.3-times and 1.2-times greater, respectively, than in intestinal epithelial cell mitochondria. As a percentage of total tissue protein, however, liver homogenates contain 10-20 times more CPS and 5-10 times more OCT than is found in intestinal mucosa. These relatively large differences in enzyme protein levels between the two tissues are not reflected by differences in their mRNA levels. As a percentage of total translational activity in vitro (based on incorporation of [35S]methionine), total liver mRNA directed synthesis of about twice as much precursor CPS (pCPS) and precursor OCT (pOCT) than did equivalent amounts of mRNA from intestinal mucosa. The ratio of pCPS and pOCT mRNA levels between the two tissues (2:1, liver:intestinal mucosa) was confirmed by dot-blot and Northern hybridizations employing specific cDNA probes. The sizes of the respective mRNAs were the same for the two tissues: about 6000 residues for pCPS mRNA and about 1700 residues for pOCT mRNA.     

Dominant Expression of Rat Prostanoid DP Receptor mRNA in Leptomeninges, Inner Segments of Photoreceptor Cells, Iris Epithelium, and Ciliary Processes

Abstract: Prostaglandin (PG) D₂ is one of the major prostanoids in the mammalian brain and eye tissues. Its function is mediated by the prostanoid DP receptor, which is specific for PGD₂ among the various prostanoids. In this study, we cloned the full-length cDNA for the rat DP receptor and used it for detection of DP receptor mRNA in various rat tissues. Northern blotting and RT-PCR analyses revealed that this DP receptor was expressed most intensely in the eye tissues, moderately in the leptomeninges and oviduct, and weakly in the epididymis. The tissue distribution profile of the mRNA for the rat DP receptor is overlapped with those of hematopoietic and lipocalin-type PGD synthases. Among rat eye tissues, the expression was the highest in the iris. In situ hybridization and in situ RT-PCR revealed DP receptor mRNA to be localized in the epithelium of the iris and ciliary body and in photoreceptor cells of the retina, suggesting the involvement of the receptor in the physiological regulation of intraocular pressure and the vision process. In the brain, DP receptor mRNA was dominantly expressed in the leptomeninges and was not detected in the brain parenchyma including the ventral rostral forebrain, the surface area of which is reportedly involved in sleep induction by PGD₂.     

A novel osteoblast-derived C-type lectin that inhibits osteoclast formation

We have cloned and expressed murine osteoclast inhibitory lectin (mOCIL), a 207-amino acid type II transmembrane C-type lectin. In osteoclast formation assays of primary murine calvarial osteoblasts with bone marrow cells, antisense oligonucleotides for mOCIL increased tartrate-resistant acid phosphatase-positive mononucleate cell formation by 3-5-fold, whereas control oligonucleotides had no effect. The extracellular domain of mOCIL, expressed as a recombinant protein in Escherichia coli, dose-dependently inhibited multinucleate osteoclast formation in murine osteoblast and spleen cell co-cultures as well as in spleen cell cultures treated with RANKL and macrophage colony-stimulating factor. Furthermore, mOCIL acted directly on macrophage/monocyte cells as evidenced by its inhibitory action on adherent spleen cell cultures, which were depleted of stromal and lymphocytic cells. mOCIL completely inhibited osteoclast formation during the proliferative phase of osteoclast formation and resulted in 70% inhibition during the differentiation phase. Osteoblast OCIL mRNA expression was enhanced by parathyroid hormone, calcitriol, interleukin-1alpha and -11, and retinoic acid. In rodent tissues, Northern blotting, in situ hybridization, and immunohistochemistry demonstrated OCIL expression in osteoblasts and chondrocytes as well as in a variety of extraskeletal tissues. The overlapping tissue distribution of OCIL mRNA and protein with that of RANKL strongly suggests an interaction between these molecules in the skeleton and in extraskeletal tissues.     

Identification of xanthine dehydrogenase/xanthine oxidase as a rat Paneth cell zinc-binding protein

Paneth cells are zinc-containing cells localized in small intestinal crypts, but their function has not been fully elucidated. Previously, we showed that an intravenous injection of diphenylthiocarbazone (dithizone), a zinc chelator, induced selective killing of Paneth cells, and purified a zinc-binding protein in Paneth cells. In the present study, we further characterized one of these proteins, named zinc-binding protein of Paneth cells (ZBPP)-1. Partial amino acid sequences of ZBPP-1 showed identity with rat xanthine dehydrogenase (XD)/xanthine oxidase (XO). Anti-rat XD antibody (Ab) recognized ZBPP-1, and conversely anti ZBPP-1 Ab recognized 85 kDa fragment of rat XD in Western blotting. Messenger RNA and protein levels of XD were consistent with our previous data on the fluctuation of Paneth cell population after dithizone injection. Thus, ZBPP-1 is an 85 kDa fragment of XD/XO in Paneth cells. XD/XO in Paneth cells may play important roles in intestinal function.     

A role for membrane-type serine protease (MT-SP1) in intestinal epithelial turnover

Membrane type-serine protease 1 (MT-SP1) plays potential roles in the process of invasion and metastasis of carcinomas. In the present study, we cloned a rat MT-SP1 cDNA and investigated the intestinal distribution and proteolytic properties of the enzyme. By in situ hybridization we found the prominent expression of the mRNA in the epithelial layer of the small intestinal upper villi and of the colon, where cells are loosely attached to the basement membrane. When MT-SP1 was expressed in Caco-2, a colonic carcinoma cell line, the protein was localized exclusively on the basolateral side. A secreted form of the enzyme produced in COS-1 cells digested fibronectin and laminin. These findings suggest that MT-SP1 participates in the control of intestinal epithelial turnover by regulating the cell-substratum adhesion.     

Effect of GLP-2 on mucosal morphology and SGLT1 expression in tissue-engineered neointestine

Using tissue-engineering techniques, we have developed a neointestine that regenerates the structural and dynamic features of native small intestine. In this study, we tested neointestinal responsiveness to glucagon-like peptide 2 (GLP-2). Neointestinal cysts were engineered by seeding biodegradable polymers with neonatal rat intestinal organoid units. The cysts were matured and anastomosed to the native jejunum of syngeneic adult recipients. Animals were treated with GLP-2 [Gly2] (twice daily, 1 microg/g body wt) or vehicle alone (control) for 10 days. Rats were then killed, and tissues were harvested for analysis. Na+-glucose cotransporter (SGLT1) mRNA expression was assessed with Northern blotting and in situ hybridization. SGLT1 protein was localized by using immunofluorescence. GLP-2 administration resulted in 1.8- and 1.7-fold increases (P < 0.05) in neointestinal villus height and crypt depth, respectively. GLP-2 administration also resulted in a 2.4-fold increase (P < 0.01) in neomucosal SGLT1 mRNA expression. SGLT1 mRNA expression was localized to enterocytes throughout the villi, and SGLT1 protein was localized to the brush border of enterocytes along the entire length of villi from the neointestine of GLP-2-treated animals. The response of tissue-engineered neointestine to exogenous GLP-2 includes mucosal growth and enhanced SGLT1 expression. Therefore, tissue-engineering principles may help in dissecting the regulatory mechanisms mediating complex processes in the intestinal epithelium.     

A novel WD40 repeat protein, WDC146, highly expressed during spermatogenesis in a stage-specific manner

We have cloned a novel cDNA encoding a protein with eight WD repeat motifs and a domain similar to collagen. As the predicted size of the protein was 146 kDa, the gene was named WDC146. Here, we characterized the genomic structure, gene products, and the expression profiles. The human WDC146 gene had 22 exons spanning over 105 kb, and these exons were distributed in three islands intervened by two long introns of around 40 kb. A minimum promoter region was identified within a 0.5 kb 5'-upstream region of exon 1. WDC146 mRNA was most highly expressed in human testis on Northern blot analysis. In mouse tissues, the highest expression was also observed in testis. By in situ hybridization on rat tissues, WDC146 mRNA was detected preferentially in the pachytene stage of spermatocytes in testis, and weakly in white pulp/ marginal band of spleen and in cortex of thymus. WDC146 protein was found to be localized in nucleus. These data implied that WDC146 protein may play important roles in the mechanisms of cytodifferentiation and/or DNA recombination.     

Calcyclin (S100A6) binding protein (CacyBP) is highly expressed in brain neurons.

The expression of a novel calcyclin (S100A6) binding protein (CacyBP) in different rat tissues was determined by Western and Northern blotting. Polyclonal antibodies against recombinant CacyBP purified from E. coli exhibited the highest reaction in the brain and weaker reaction in liver, spleen, and stomach. CacyBP immunoreactivity was also detected in lung and kidney. Densitometric analysis showed that the concentration of CacyBP in the soluble fractions of total brain and cerebellum is approximately 0.17 and 0. 34 ng/microg protein, respectively. Northern blotting with a specific cDNA probe confirmed the high level of CacyBP expression in the rat brain and lower levels in other tissues examined. Immunohistochemistry and in situ hybridization of rat brain sections revealed strong expression of CacyBP in neurons of the cerebellum, hippocampus, and cortex. The in situ hybridization detected CacyBP in hippocampus as early as P7 (postnatal day 7) and a peak of expression at P21, and the expression signal was preserved until adulthood. In the entorhinal cortex, the peak of expression was observed at P7, whereas in the cerebellum it was seen at P21. The results presented here show that CacyBP is predominantly a neuronal protein. (J Histochem Cytochem 48:1195-1202)     

Extrahepatic synthesis of apolipoprotein E

Apolipoprotein E (apoE) synthesis has been examined in rat and guinea pig tissues using in vitro translation and [35S]methionine labeling of tissue slices. A number of tissues not involved in lipoprotein synthesis synthesize a protein very similar to apoE, including the spleen, adrenal, kidney, testis, ovary, heart, and lung. Although the intestine is involved in lipoprotein synthesis, apoE synthesis could not be detected in intestinal mucosa. The protein synthesized by the extrahepatic tissues was identified as apoE by its electrophoretic mobility, its immunologic reactivity with a monospecific antibody and by limited proteolysis mapping with Staphylococcus aureus V8 protease. ApoE represented between 0.02 and 0.7% of the total protein synthesized in the extrahepatic tissues, indicating that apoE mRNA is a fairly abundant mRNA in these tissues. ApoE mRNA was also detected by hybridization with a rat apoE cDNA clone, which hybridized to a single mRNA 1250 nucleotides in length in rat liver and in extrahepatic tissues. Hybridization of the apoE clone to rat genomic DNA demonstrated that the apoE gene was more heavily methylated in intestinal mucosa, which did not synthesize apoE, than in liver, testis, or kidney. 35S labeling of peritoneal macrophages revealed that both rat and guinea pig macrophages synthesized and secreted apoE in vitro. Rhesus aortic smooth muscle cells also synthesized and secreted apoE. The possible functions of apoE synthesized in the peripheral tissues are considered.     

Molecular cloning and biological activity of a novel Ha-Ras suppressor gene predominantly expressed in skeletal muscle, heart, brain, and bone marrow by differential display using clonal mouse EC cells, ATDC5.

We cloned a cDNA encoding a novel mouse protein, named A-C1, by differential display between two mouse cell lines: embryonic fibroblast C3H10T1/2 and chondrogenic ATDC5. The deduced amino acid sequence of A-C1 consists of 167 amino acids and shows 46% identity with that of a ras-responsive gene, rat Ha-rev107. Northern blot analysis showed a distinct hybridization band of 3.2 kilobases. Expression of A-C1 mRNA was detected in undifferentiated ATDC5 cells and myoblastic C2C12 cells, while none of C3H10T1/2 cells, NIH3T3 fibroblasts, Balb/c 3T3 fibroblasts, osteoblastic MC3T3-E1 cells, and ST2 bone marrow stromal cells expressed A-C1 mRNA in vitro. Moreover, A-C1 mRNA was expressed in skeletal muscle, heart, brain, and bone marrow in adult mice. By in situ hybridization, A-C1 gene expression was localized in hippocampus as well as bone marrow cells. By immunocytochemistry, A-C1 protein was detected in the cytoplasm as well as perinuclear region of the cells. Transfection of A-C1 cDNA into Ha-ras-transformed NIH3T3 cell line caused increase in the number of flat colonies and inhibition of cell growth. Our data indicate that A-C1 is expressed in some specific tissues in vivo and modulates Ha-ras-mediated signaling pathway.     

Induction of Src-suppressed C kinase substrate (SSeCKS) in vascular endothelial cells by bacterial lipopolysaccharide.

We isolated cDNA of the mouse homologue of the src-suppressed C kinase substrate (SSeCKS) and analyzed the effects of lipopolysaccharide (LPS) injection on the tissue expression pattern of this protein. Northern blotting analysis showed that SSeCKS mRNA was expressed abundantly in the testis but at undetectable levels in other tissues of untreated control mice. Intraperitoneal administration of LPS strongly induced SSeCKS mRNA expression in the lung, heart, liver, spleen, kidney, lymph node, adrenal gland, and pituitary gland, as well as in the brain. In lung and spleen, the SSeCKS mRNA levels increased almost 10-fold at 1 hr after LPS injection and persisted at high levels until 4 hr. Both in situ hybridization and immunohistochemical studies revealed that LPS administration conspicuously elevated expression of SSeCKS mRNA and protein in vascular endothelial cells of several organs. Ectopic expression of SSeCKS caused loss of cytoplasmic F-actin fibers in the mouse endothelial cell line LEII. These results indicate that SSeCKS is one of the major LPS-responsive proteins and may participate in alteration of cytoskeletal architecture in endothelial cells during inflammation.     

Calreticulin--an endoplasmic reticulum protein with calcium-binding activity is also found in the extracellular matrix.

Previous studies have reported that calreticulin (CRT), a calcium-binding and chaperoning protein, is expressed only in the endoplasmatic reticulum, nucleus and at the cell surface. In this study we clearly show that odontoblasts and predentin matrix contain CRT. To our knowledge, this is the first time CRT has been described in the extracellular matrix. The expression of CRT was studied by immunohistochemistry, ultrastructural immunocytochemistry and in situ hybridization in developing rat teeth. CRT was detected as a 59-kDa protein in rat pulp cell culture medium and dentin extracellular matrix extract by Western blotting. The presence of the protein was shown in rat odontoblasts and predentin with immunohistochemistry. At the ultrastructural level, the labeling was distributed in the rat odontoblasts, ameloblasts and predentin. Northern blotting showed the presence of CRT mRNA in rat molars, which was confirmed by in situ hybridization in odontoblasts and ameloblasts. We now present the first convincing evidence that CRT is found in extracellular matrix where it may play an important role in mineralization.