CD44 Deficiency Is Associated with Increased Susceptibility to Stress-Induced Anxiety-like Behavior in Mice.

CD44 is a cell surface adhesion molecule and its principal ligand is hyaluronic acid (HA), a key component of the brain's extracellular matrix. CD44 levels are decreased in the cerebrospinal fluid (CSF) of depressed individuals, and the CD44 gene has been identified in genome wide association study as a possible risk gene in suicidal behavior. In order to define the pathobiological mechanisms by which CD44 may affect behavior, we investigated the role of CD44 using male CD44 knockout (CD44KO) and wild-type mice that underwent chronic mild stress (CMS). Behavior was characterized using the sucrose preference and forced swim tests, open field, novel object recognition, social preference, and the elevated plus maze tests. Gene expression in hippocampus was evaluated using quantitative real-time PCR. Brain monoamines and their metabolites were assessed by high-performance liquid chromatography and serum HA and IL-1ß levels were measured using ELISA and electrochemiluminescence assays. CD44KO mice were more susceptible to stress-induced anxiety-like behavior and displayed increased anhedonia and despair than the wild-type controls. The behavioral phenotype of stressed CD44KO mice was associated with reduced cortical serotonergic and striatal dopaminergic turnover. The hippocampal expression of the receptor for HA-mediated motility (RHAMM) was reduced in the non- stressed CD44KO mice compared with WT mice, in a value similar to that observed in WT mice following exposure to stress. Taken together, our experiments suggest that CD44 plays a key role in stress response in mice.

Mitogenic G(i) protein-MAP kinase signaling cascade in MC3T3-E1 osteogenic cells: activation by C-terminal pentapeptide of osteogenic growth peptide [OGP(10-14)] and attenuation of activation by cAMP.

In osteogenic and other cells the mitogen-activated protein (MAP) kinases have a key role in regulating proliferation and differentiated functions. The osteogenic growth peptide (OGP) is a 14 mer mitogen of osteogenic and fibroblastic cells that regulates bone turnover, fracture healing, and hematopoiesis, including the engraftment of bone marrow transplants. It is present in the serum and extracellular fluid either free or complexed to OGP-binding proteins (OGPBPs). The free immunoreactive OGP consists of the full length peptide and its C-terminal pentapeptide OGP(10-14). In the present study, designed to probe the signaling pathways triggered by OGP, we demonstrate in osteogenic MC3T3 E1 cells that mitogenic doses of OGP(10-14), but not OGP, enhance MAP kinase activity in a time-dependent manner. The OGP(10-14)-induced stimulation of both MAP kinase activity and DNA synthesis were abrogated by pertusis toxin, a G(i) protein inhibitor. These data offer direct evidence for the occurrence in osteogenic cells of a peptide-activated, mitogenic Gi protein-MAP kinase-signaling cascade. Forskolin and dBu(2)-cAMP abrogated the OGP(10-14)-stimulated proliferation, but induced only 50% inhibition of the OGP(10-14)-mediated MAP kinase activation, suggesting additional MAP kinase-dependent, OGP(10-14)-regulated, cellular functions. Finally, it is demonstrated that OGP(10-14) is the active form of OGP, apparently generated proteolytically in the extracellular milieu upon dissociation of OGP-OGPBP complexes.

Structure-bioactivity of C-terminal pentapeptide of osteogenic growth peptide [OGP(10-14)].

The amino acid sequence of osteogenic growth peptide (OGP) consists of 14 residues identical to the C-terminal tail of histone H4. Native and synthetic OGP are mitogenic to osteoblastic and fibroblastic cells and enhance osteogenesis and hematopoiesis in vivo. The C-terminal truncated pentapeptide of OGP, H-Tyr-Gly-Phe-Gly-Gly-OH [OGP(10-14)], is a naturally occurring osteoblastic mitogen, equipotent to OGP. The present study assesses the role of individual amino acid residues and side chains in the OGP(10-14) mitogenic activity which showed a very high correlation between osteoblastic and fibroblastic cell cultures. Truncation of either Tyr10 or its replacement by Ala or D-Ala resulted in substantial, but not complete, loss of activity. Nevertheless, only a small loss of activity was observed following removal of the Tyr10 amino group. No further loss occurred consequent to the monoiodination of desaminoTyr10 on meta-position. However, a marked decrease in proliferative activity followed removal of the Tyr10 phenolic or the Phe12 aromatic group. Loss of activity of a similar magnitude also occurred subsequent to replacing Gly11 with L- or D-Ala. Approximately 50% loss of mitogenic activity occurred subsequent to truncation of Gly14 or blocking the C-terminal group as the methyl ester. All other modifications of the C-terminus and L- or D-Ala substitution of Gly13 resulted in 70-97% decrease in activity. Collectively, these data suggest that the integrity of the pharmacophores presented by Tyr and Phe side chains, as well as the Gly residues at the C-terminus, are important for optimal bioactivity of OGP(10-14).

Isolation of mitogenically active C-terminal truncated pentapeptide of osteogenic growth peptide from human plasma and culture medium of murine osteoblastic cells.

The osteogenic growth peptide (OGP) is a 14-amino acid stromal cell mitogen that stimulates in vivo osteogenesis and hematopoiesis. In the blood circulation and cell culture conditioned medium immunoreactive OGP (irOGP), identified using antibodies raised against the OGP C-terminal region, presents free and bound forms. The bound form consists entirely of the full length peptide. The present study was designed to investigate the identity of free irOGP under nondenaturing conditions. Fresh human serum and culture medium conditioned with murine osteoblastic MC3T3 E1 cells were fractionated using ultrafiltration (3000 molecular weight cut-off). Hydrophobic chromatography of the ultrafiltrate, immunoscreening of chromatographic fractions with antibodies directed against the OGP C-terminal region and amino acid sequencing of immunoreactive peaks demonstrated the presence of two mitogens, the full length OGP and a C-terminal truncated form, OGP(10-14). The OGP(10-14) derived from both serum and conditioned medium, as well as the synthetic pentapeptide [sOGP(10-14)], shared the in vitro OGP proliferative activity. However, in a competitive binding assay, devised to assess the OGP-OGP binding protein (OGPBP) complex formation, sOGP(10-14) failed to compete out radiolabeled OGP from the complex. It is concluded that OGP(10-14) is a naturally occurring human and murine mitogen. In addition, the data suggests that the OGP(10-14) is generated from OGP by proteolytic cleavage upon dissociation of the OGP-OGPBP complexes.

Biosynthesis of osteogenic growth peptide via alternative translational initiation at AUG85 of histone H4 mRNA.

The osteogenic growth peptide (OGP) is an extracellular mitogen identical to the histone H4 (H4) COOH-terminal residues 90-103, which regulates osteogenesis and hematopoiesis. By Northern analysis, OGP mRNA is indistinguishable from H4 mRNA. Indeed, cells transfected with a construct encoding [His102]H4 secreted the corresponding [His13]OGP. These results suggest production of OGP from H4 genes. Cells transfected with H4-chloramphenicol acetyltransferase (CAT) fusion genes expressed both "long" and "short" CAT proteins. The short CAT was retained following an ATG --> TTG mutation of the H4 ATG initiation codon, but not following mutation of the in-frame internal ATG85 codon, which, unlike ATG1, resides within a perfect context for translational initiation. These results suggest that a PreOGP is translated starting at AUG85. The translational initiation at AUG85 could be inhibited by optimizing the nucleotide sequence surrounding ATG1 to maximally support upstream translational initiation, thus implicating leaky ribosomal scanning in usage of the internal AUG. Conversion of the predicted PreOGP to OGP was shown in a cell lysate system using synthetic [His102]H4-(85-103) as substrate. Together, our results demonstrate that H4 gene expression diverges at the translational level into the simultaneous parallel production of both H4, a nuclear structural protein, and OGP, an extracellular regulatory peptide.

Human alpha 2-macroglobulin is an osteogenic growth peptide-binding protein.

The osteogenic growth peptide (OGP) is a 14mer mitogen of osteoblastic and fibroblastic cells. Physiologically, OGP is present in high abundance in human and other mammalian sera. Most of the serum OGP is complexed noncovalently to heat sensitive, high molecular weight OGP-binding proteins (OGPBPs). Changes in serum OGP levels that follow bone marrow ablation and the low doses of exogenous OGP required for the stimulation of bone formation suggest a regulatory role for the OGPBPs. In the present work, the OGP binding activity was monitored by competitive binding to [3-125I(Tyr10)]-sOGP and the corresponding complexes were demonstrated on nondenaturing cathodic polyacrylamide gel electrophoresis. We show that OGP binds to both native and activated human plasma alpha 2-macroglobulin (alpha 2M). alpha 2M was also immunoidentified in reduced and nonreduced SDS-polyacrylamide gel electrophoresis of OGP-affinity purified plasma-derived proteins. Immunoreactive OGP was detected in commercial preparations of both forms of alpha 2M; OGP was purified to homogeneity from the commercial preparation of activated alpha 2M. In MC3T3 E1 cells, native alpha 2M, at concentrations < 50 ng/mL, had a substantially increased mitogenic effect in the presence of synthetic, native-like, OGP (sOGP). Similar amounts of activated alpha 2M inhibited the sOGP proliferative effect. These results suggest that the native alpha 2M enhances the immediate availability of OGP to its target cells. Activated alpha 2M may participate in the removal of OGP from the system.

Isolation of osteogenic growth peptide from osteoblastic MC3T3 E1 cell cultures and demonstration of osteogenic growth peptide binding proteins.

The osteogenic growth peptide (OGP) was recently characterized in regenerating bone marrow. In experimental animals in increases osteogenesis and hemopoiesis. In stromal cell cultures OGP stimulates proliferation, alkaline phosphatase activity, and matrix mineralization. OGP in high abundance is present in normal human and animal serum mainly complexed to OGP binding protein (OGPBP) or proteins. Here we show the presence of two OGPBPs, OGPBP-1, and OGPBP-2, in cultures of osteoblastic MC3T3 E1 cells. Immunoreactive OGP (irOGP) also accumulates in the medium of these cultures and in cultures of NIH 3T3 fibroblasts. A large amount of irOGP was released by heat inactivation of OGPBP-2 and purified by ultrafiltration and hydrophobic HPLC. The purified irOGP was identical to OGP obtained previously from rat regenerating bone marrow and human serum in terms of its amino acid sequence, immunoreactivity, and mitogenicity. Osteoblastic and fibroblastic cell proliferation can be arrested by anti-OGP antibodies and rescued by exogenous OGP, indicating that in the absence of serum or other exogenous growth stimulators the endogenously produced OGP is both necessary and sufficient for baseline proliferation. The OGP production is up- and down-regulated, respectively, by low and high doses of exogenous OGP in a manner consistent with an autoregulated feedback mechanism. The most effective OGP dose in MC3T3 E1 cells is at least two orders of magnitude lower than that in non-osteoblastic cell systems. This differential sensitivity of the osteoblastic cells could result in a preferential anabolic effect of OGP in bone.

Fanconi anemia revisited: old ideas and new advances.

This review summarizes both historical and more recent data on the clinical, cellular and genetic features of Fanconi anemia (FA), a rare autosomal recessive disorder. FA patients are characterized by pancytopenia, congenital malformations, growth delay and an increased susceptibility to the development of malignancies, particularly acute myelogenous leukemia. FA cells show chromosomal fragility, slow growth and increased sensitivity to DNA crosslinking agents. FA can be caused by defects in any one of at least four genes. Two general hypotheses have been proposed to explain the underlying defect: loss of a DNA repair function or of a step in the defense toward oxygen toxicity. After many attempts to clone the FA genes, the first one, that defective in group C, has been cloned by complementation of the increased sensitivity of FA(C) cells to mitomycin C and diepoxybutane. This gene (FACC) codes for a novel protein and is ubiquitously expressed. Mutations in various FA(C) patients that cause loss of function have been identified. The review concludes by suggesting directions for future research in FA.

A Leu554-to-Pro substitution completely abolishes the functional complementing activity of the Fanconi anemia (FACC) protein.

Three cDNA transcripts corresponding to complementation group C of Fanconi anemia (FA) were recently cloned. We confirm that the correct reading frame was reported and that a protein of an apparent molecular mass of 60 kDa is translated. A T-to-C transition at base 1,661 in the open reading frame is the only change found to date in the FA(C) cell line, resulting in a codon substitution from leucine554 to proline. Using site directed in vitro mutagenesis, we demonstrate that this mutation completely abolishes the activity of the FACC protein as analyzed by functional complementation assay. The physiological significance of this mutation is thus confirmed.

Cloning of cDNAs for Fanconi's anaemia by functional complementation.

Fanconi's anaemia is a rare autosomal recessive disorder characterized by progressive pancytopaenia and a cellular hypersensitivity to DNA crosslinking agents. Four genetic complementation groups have been identified so far, and here we use a functional complementation method to clone complementary DNAs that correct the defect of group C cells. The cDNAs encode alternatively processed transcripts of a new gene, designated FACC, which is mutated in group C patients. The predicted FACC polypeptide does not contain any motifs common to other proteins and so represents a new gene involved in the cellular response to DNA damage.

Suppression of somatic embryogenesis in Citrus cell cultures by extracellular proteins.

Nucellar-derived cell cultures of sour orange (Citrus aurantium L.) proliferate as proembryogenic masses. By a change in the carbon source of the medium from sucrose to glycerol they are induced to undergo synchronous embryogenesis forming embryo initials that develop into globular embryos. The proembryogenic masses released glycoproteins to the medium. Exogenous addition of the glycoproteins to cells in glycerol-containing medium modified the course of embryo development in a dose-dependent manner. Addition of 20 µg · ml(-1) of glycoproteins blocked embryogenesis and resulted in an accumulation of embryo initials. When glycoproteins were added to cultures containing advanced globularstage embryos further development was suppressed. The inhibitory component of the glycoproteins was found to be a family of polypeptides with apparent molecular masses of 53-57 kDa. While these proteins normally accumulated only in cultures of proembryogenic masses, they could be induced to accumulate in glycerol-containing medium by the addition of the glycoproteins. Thus, their accumulation was not a direct consequence of the type of growth medium used or the developmental state of the cultures. The results indicate that the 53-to 57 kDa glycoproteins could play a regulatory role in in-vitro embryogenesis in sour orange. The normal progression of embryo development appears to depend, in an obligatory manner, on the absence of these glycosylated extracellular proteins from the medium.

Production of flavanone-neohesperidosides in Citrus embryos.

Grapefruit (Citrus paradisi) tissue cultures were examined for qualitative and quantitative changes in flavanone-neohesperidoside content during somatic embryogenesis. Embryos cultured in vitro contain naringin and a rhamnosyl-transferase activity which is capable of rhamnosylating position 2 on the flavanone glucosides. Rhamnosylation is carried out only in embryos cultivated on solid medium but not in embryos grown in suspension cell cultures.