Murine p38-delta mitogen-activated protein kinase, a developmentally regulated protein kinase that is activated by stress and proinflammatory cytokines

The p38 mitogen-activated protein kinases (MAPK) play a crucial role in stress and inflammatory responses and are also involved in activation of the human immunodeficiency virus gene expression. We have isolated the murine cDNA clones encoding p38-delta MAPK, and we have localized the p38-delta gene to mouse chromosome 17A3-B and human chromosome 6p21.3. By using Northern and in situ hybridization, we have examined the expression of p38-delta in the mouse adult tissues and embryos. p38-delta was expressed primarily in the lung, testis, kidney, and gut epithelium in the adult tissues. Although p38-delta was expressed predominantly in the developing gut and the septum transversum in the mouse embryo at 9.5 days, its expression began to be expanded to many specific tissues in the 12.5-day embryo. At 15.5 days, p38-delta was expressed virtually in most developing epithelia in embryos, suggesting that p38-delta is a developmentally regulated MAPK. Interestingly, p38-delta and p38-alpha were similar serine/threonine kinases but differed in substrate specificity. Overall, p38-delta resembles p38-gamma, whereas p38-beta resembles p38-alpha. Moreover, p38-delta is activated by environmental stress, extracellular stimulants, and MAPK kinase-3, -4, -6, and -7, suggesting that p38-delta is a unique stress-responsive protein kinase.     

Pattern of expression of HtrA1 during mouse development.

The human HtrA family of proteases consists of four members: HtrA1, HtrA2, HtrA3, and HtrA4. In humans the four HtrA homologues appear to be involved in several important functions such as cell growth, apoptosis, and inflammatory reactions, and they control cell fate via regulated protein metabolism. In previous studies it was shown that the expression of HtrA1 was ubiquitous in normal adult human tissues. Here we examined the expression of HtrA1 protein and its corresponding mRNA during mouse embryogenesis using Northern blotting hybridization, RT-PCR, and immunohistochemical staining analyses. Our results indicate that HtrA1 is expressed in a variety of tissues in mouse embryos. Furthermore, this expression is regulated in a spatial and temporal manner. Relatively low levels of HtrA1 mRNA are detected in embryos at the beginning of organogenesis (E8), and the levels of expression increase during late organogenesis (E14-E19). Our results show that HtrA1 was expressed during embryonic development in specific areas where signaling by TGFbeta family proteins plays important regulatory roles. The expression of HtrA1, documented both at mRNA and protein levels by RT-PCR and immunohistochemistry in the developing nervous system, is consistent with a possible role of this protein both in dividing and postmitotic neurons, possibly via its documented inhibitory effects on TGFbeta proteins. An exhaustive knowledge of the different cell- and tissue-specific patterns of expression of HtrA1 in normal mouse embryos is essential for a critical evaluation of the exact role played by this protein during development.     

Comparison of expression patterns and cell adhesion properties of the mouse biliary glycoproteins Bbgp1 and Bbgp2.

Biliary glycoproteins are members of the carcinoembryonic antigen (CEA) family and behave as cell adhesion molecules. The mouse genome contains two very similar Bgp genes, Bgp1 and Bgp2, whereas the human and rat genomes contain only one BGP gene. A Bgp2 isoform was previously identified as an alternative receptor for the mouse coronavirus mouse hepatitis virus. This isoform consists of two extracellular immunoglobulin domains, a transmembrane domain and a cytoplasmic tail of five amino acids. In this report, we have examined whether the Bgp2 gene can express other isoforms in different mouse tissues. We found only one other isoform, which has a long cytoplasmic tail of 73 amino acids. The long cytodomain of the Bgp2 protein is highly similar to that of the Bgp1/4L isoform. The Bgp2 protein is expressed in low amounts in kidney and in a rectal carcinoma cell line. Antibodies specific to Bgp2 detected a 42-kDa protein, which is expressed at the cell surface of these samples. Bgp2 was found by immunocytochemistry in smooth muscle layers of the kidney, the uterus, in gut mononuclear cells and in the crypt epithelia of intestinal tissues. Transfection studies showed that, in contrast with Bgp1, the Bgp2 glycoprotein was not directly involved in intercellular adhesion. However, this protein is found in the proliferative compartment of the intestinal crypts and in cells involved in immune recognition. This suggests that the Bgp2 protein represents a distinctive member of the CEA family; its unusual expression patterns in mouse tissues and the unique functions it may be fulfilling may provide novel clues about the multiple functions mediated by a common BGP protein in humans and rats.     

The CEA family: a system in transitional evolution?

The CEA family consists of two structurally and functionally distinct sub-groups; the group including CEA, NCA and CGM-6 which are cell surface-bound by phosphatidyl-inositol (PI) linkages, and the group of BGP splice variants which have trans-membrane and cytoplasmic domains. Although all CEA family members mediate intercellular adhesion in vitro, the PI-linked group show Ca++ and temperature independent adhesion whereas the BGP group show rapidly reversible Ca++ and temperature dependent adhesion. From the close alignment in cDNA nucleotide sequences between family members and between repeated domains in one family member, it is apparent that the CEA family is now rapidly evolving; in fact, analogs of only the trans-membrane BGP group have been found so far in the mouse. The addition of a new group of potent adhesion molecules to complex species at some time after the rodent radiation has strong evolutional implications, which are discussed.     

Lipase H,a new member of the triglyceride lipase family synthesized by the intestine

We report here the molecular cloning of a novel member of the triglyceride lipase family, a 2.4-kb cDNA encoding human lipase H (LIPH) and the mouse ortholog (Liph). The human LIPH cDNA encodes a 451-amino-acid protein with a lipase domain. Mouse Liph shows 85% amino acid identity and 75% nucleotide identity to human LIPH. Human LIPH exhibits 47% identity with phosphatidylserine-specific phospholipase A1 (PS-PLA1) and 46% identity with endothelial lipase (LIPG) and lipoprotein lipase (LPL). LIPH is localized on human chromosome 3q27-q28. Northern blot analysis revealed specific expression of LIPH mRNA in intestine, lung, and pancreas. Lipase H protein was also detected in human intestine. Lipase H is a secreted protein with an apparent molecular weight of 63 kDa. Although several lipid substrates were tested, the lipid substrate of LIPG was not identified. Like the other members of this gene family, LIPH may be involved in lipid and energy metabolism.     

EHD1--an EH-domain-containing protein with a specific expression pattern.

A cDNA that is a member of the eps15 homology (EH)-domain-containing family and is expressed differentially in testis was isolated from mouse and human. The corresponding genes map to the centromeric region of mouse chromosome 19 and to the region of conserved synteny on human chromosome 11q13. Northern analysis revealed two RNA species in mouse. In addition to the high levels in testis, expression was noted in kidney, heart, intestine, and brain. In human, three RNA species were evident. The smaller one was predominant in testis, while the largest species was evident in other tissues as well. The predicted protein sequence has an EH domain at its C-terminus, including an EF, a Ca2+ binding motif, and a central coiled-coil structure, as well as a nucleotide binding consensus site at its N-terminus. As such, it is a member of the EH-domain-containing protein family and was designated EHD1 (EH domain-containing 1). In cells in tissue culture, we localized EHD1 as a green fluorescent protein fusion protein, in transferrin-containing, endocytic vesicles. Immunostaining of different adult mouse organs revealed major expression of EHD1 in germ cells in meiosis, in the testes, in adipocytes, and in specific retinal layers. Results of in situ hybridization to whole embryos and immunohistochemical analyses indicated that EHD1 expression was already noted at day 9.5 in the limb buds and pharyngeal arches and at day 10.5 in sclerotomes, at various elements of the branchial apparatus (mandible and hyoid), and in the occipital region. At day 15.5 EHD1 expression peaked in cartilage, preceding hypertrophy and ossification, and at day 17.5 there was no expression in the bones. The EHD1 gene is highly conserved between nematode, Drosophila, mouse, and human. Its predicted protein structure and cellular localization point to the possibility that EHD1 participates in ligand-induced endocytosis.     

Isolation and initial characterization of the mouse Dnmt3l gene

We have isolated and sequenced the mouse zinc finger gene, Dnmt3l (DNA cytosine-5-methyltransferase 3-like), on mouse chromosome 10, showing similarity to members of the DNMT3/Dnmt3 family. The Dnmt3l protein contains an ADD zinc finger, which Dnmt3l shares with other Dnmt3 family members and Atrx. RT-PCR analysis showed Dnmt3l expression in testis, thymus, ovary, and heart, as well as in 7-day, 15-day, and 17-day mouse embryos.     

Evidence for differential functions of the p50 and p65 subunits of NF-kappa B with a cell adhesion model.

The p50 and p65 subunits of NF-kappa B represent two members of a gene family that shares considerable homology to the rel oncogene. Proteins encoded by these genes form homo- and heterodimers which recognize a common DNA sequence motif. Recent data have suggested that homodimers of individual subunits of NF-kappa B can selectively activate gene expression in vitro. To explore this possibility in a more physiological manner, murine embryonic stem (ES) cells were treated with phosphorothio antisense oligonucleotides to either p50 or p65. Within 5 h after exposure to phosphorothio antisense p65 oligonucleotides, cells exhibited dramatic alterations in adhesion properties. Similar findings were obtained in a stable cell line that expressed a dexamethasone-inducible antisense mRNA to p65. Although antisense oligonucleotides raised against both p50 and p65 elicited a significant reduction in their respective mRNAs, only the cells treated with antisense p50 maintained a normal morphology. However, 6 days following removal of leukemia-inhibiting factor, a growth factor which suppresses embryonic stem cell differentiation, adhesion properties of cells treated with the antisense p50 oligonucleotides were markedly affected. The ability of the individual antisense oligonucleotides to elicit differential effects on cell adhesion, a property dependent upon the stage of differentiation, suggests that the p50 and p65 subunits of NF-kappa B regulate gene expression either as homodimers or as heterodimers with other rel family members. Furthermore, the finding that reduction in p65 expression alone had profound effects on cell adhesion properties indicates that p65 plays an important role in nonstimulated cells and cannot exist solely complexed with the cytosolic inhibitory protein I kappa B.     

Characterization and differential expression of a human gene family of olfactomedin-related proteins

Olfactomedin-related proteins are secreted glycoproteins with conserved C-terminal motifs. Olfactomedin was originally identified as the major component of the mucus layer that surrounds the chemosensory dendrites of olfactory neurons. Homologues were subsequently found also in other tissues, including the brain and in species ranging from Caenorhabditis elegans to Homo sapiens. Most importantly, the TIGR/myocilin protein, expressed in the eye and associated with the pathogenesis of glaucoma, is an olfactomedin-related protein. The prevalence of olfactomedin-related proteins among species and their identification in different tissues prompted us to investigate whether a gene family exists within a species, specifically Homo sapiens. A GenBank search indeed revealed an entire human gene family of olfactomedin-related proteins with at least five members, designated hOlfA through hOlfD and the TIGR/myocilin protein. hOlfA corresponds to the rat neuronal AMZ protein. Phylogenetic analyses of 18 olfactomedin-related sequences resolved four distinct subfamilies. Among the human proteins, hOlfA and hOlfC, both expressed in brain, are most closely related. Northern blot analyses of 16 human tissues demonstrated highly specific expression patterns: hOlfA is expressed in brain, hOlfB in pancreas and prostate, hOlfC in cerebellum, hOlfD in colon, small intestine and prostate and TIGR/myocilin in heart and skeletal muscle. The link between TIGR/myocilin and ocular hypertension and the expression of several of these proteins in mucus-lined tissues suggest that they play an important role in regulating physical properties of the extracellular environment. Future studies can now assess whether other members of this gene family, like TIGR/myocilin, are also associated with human disease processes.