Expression and activity of ADAMTS-5 in synovium.

ADAMTS proteinases, belonging to the adamalysin subfamily of metalloproteinases, have been implicated in a variety of cellular events such as morphogenesis, cell migration, angiogenesis, ovulation and extracellular matrix breakdown. Aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-5) have been identified in cartilage and are largely responsible for cartilage aggrecan breakdown. We have shown previously that synovium, the membrane lining diarthrodial joints, generates soluble aggrecanase activity. We report here the expression, localization and activity of ADAMTS-5 from human arthritic and bovine synovium. ADAMTS-5 was expressed constitutively in synovium with little or no transcriptional regulation by recombinant human interleukin-1 alpha or all-trans-retinoate, factors previously shown to upregulate aggrecanase activity in cartilage. Aggrecanase activity generated by synovium in vitro and recombinant ADAMTS-5 cleaved aggrecan extensively, resulting in aggrecan fragments similar to those generated by chondrocyte-derived aggrecanases, and the activity was inhibited by heparin. ADAMTS-5 was immunolocalized in human arthritic synovium, where staining was mostly pericellular, particularly in the synovial lining and around blood vessels; some matrix staining was also seen. The possibility that synovium-derived ADAMTS-5 may play a role in cartilage aggrecan breakdown is discussed.

ADAMTS9, a novel member of the ADAM-TS/ metallospondin gene family.

ADAM-TS/metallospondin genes encode a new family of proteins with structural homology to the ADAM metalloprotease-disintegrin family. However, unlike other ADAMs, these proteins contain thrombospondin type 1 (TSP1) repeats at the carboxy-terminal end and are secreted proteins instead of being membrane bound. Members of the ADAM-TS family have been implicated in the cleavage of proteoglycans, the control of organ shape during development, and the inhibition of angiogenesis. We have cloned a new member of the ADAM-TS/metallospondin family designated here as ADAMTS9. This protein has a metalloprotease domain, a disintegrin-like domain, one internal TSP1 motif, and three carboxy-terminal TSP1-like submotifs. In contrast to other ADAM-TS family members, ADAMTS9 is expressed in all fetal tissues examined as well as some adult tissues. Using FISH and radiation hybrid analysis, we have localized ADAMTS9 to chromosome 3p14.2-p14.3, an area known to be lost in hereditary renal tumors.

The copper transport protein Atox1 promotes neuronal survival.

Atox1, a copper transport protein, was recently identified as a copper-dependent suppressor of oxidative damage in yeast lacking superoxide dismutase. We have previously reported that Atox1 in the rat brain is primarily expressed in neurons, with the highest levels in distinct neuronal subtypes that are characterized by their high levels of metal, like copper, iron, and zinc. In this report, we have transfected the Atox1 gene into several neuronal cell lines to increase the endogenous level of Atox1 expression and have demonstrated that, under conditions of serum starvation and oxidative injury, the transfected neurons are significantly protected against this stress. This level of protection is comparable with the level of protection seen with copper/zinc superoxide dismutase and the anti-apoptotic gene bcl-2 that had been similarly transfected. Furthermore, neuronal cell lines transfected with a mutant Atox1 gene, where the copper binding domain has been modified to prevent metal binding, do not afford protection against serum starvation resulting in apoptosis. Therefore, Atox1 is a component of the cellular pathways used for protection against oxidative stress.

Nephroblastoma overexpressed gene (NOV) codes for a growth factor that induces protein tyrosine phosphorylation.

NOV (nephroblastoma overexpressed gene) is a member of the CCN (connective tissue growth factor [CTGF], Cyr61/Cef10, NOV) family of proteins. These proteins are cysteine-rich and are noted for having growth-regulatory functions. We have isolated the rat NOV gene, and the DNA sequence shares 90% identity with the mouse and 80% identity with the human sequences. The rat NOV gene was expressed in all rat tissues examined, including brain, lung, heart, kidney, liver, spleen, thymus and skeletal muscle. Higher levels of rat NOV mRNA were seen in the brain, lung and skeletal muscle compared to the other tissues. Examination of NOV expression in various human cell lines revealed that NOV was expressed in U87, 293, T98G, SK-N-MC and Hs683 but not in HepG2, HL60, THP1 and Jurkat. The human NOV gene was transfected into 293 cells and the expressed protein purified. When 3T3 fibroblasts were treated with this recombinant NOV protein, a dose-dependent increase in proliferation was observed. Analysis of tyrosine-phosphorylated proteins revealed that when 3T3 cells were treated with NOV, a 221 kDa protein was phosphorylated. These data suggest that NOV can act as a growth factor for some cells and binds to a specific receptor that leads to the phosphorylation of a 221 kDa protein.

Expression profile of the copper homeostasis gene, rAtox1, in the rat brain.

In humans the regulation of cellular copper homeostasis is essential for proper organ development and function. A novel cytosolic protein, named Atox 1, was recently identified in yeast that functions in shuttling intracellular mononuclear copper [Cu(I)] to copper-requiring proteins. Atox 1 and its human homolog, hAtox1, are members of an emerging family of proteins termed copper chaperones that are involved in the maintenance of copper homeostasis. Northern blot analysis demonstrates that Atox 1 is widely expressed at varying levels in a variety of rat tissues including brain. Using in situ hybridization histochemistry, we characterized the expression profile for the rat homolog of Atox1 (rAtox1) in the normal adult rat brain. There is widespread expression within the brain that appears to be primarily neuronal. The highest levels of Atox1 message consists of distinct neuronal subtypes that are also characterized by their high levels of metals like copper, iron, and zinc, which include the pyramidal neurons of the cerebral cortex and hippocampus in addition to the neurons of the locus coeruleus. The high levels of a metal chaperone like Atox1 in subsets of neurons that also sequester metals suggests that Atox1 may be important in maintaining the functionality of metal requiring enzymes. A detailed analysis of the restricted expression profile for a novel copper chaperone, rAtox1, is described in the adult rat CNS. Further analysis shows that Atoxl expression is associated with neuronal populations that sequester copper.

Subtractive hybridization of cDNA libraries.

The technique of subtractive hybridization is used to enrich abundant cDNAs that differ between two cell populations. This approach is well suited for systems in which a homogenous cell population is activated in culture with an agent or factor that induces the transcription of genes that are either not present or at low abundance in the unactivated state. Whereas this scenario is the ideal model, models of comparison between heterogeneous cell cultures or even tissues have been analyzed by subtractive hybridization with success. The magnitude of success is directly related to the magnitude of the differential upregulation of relevant transcripts in the activated state over the unactivated state.

Murine GBP-2: a new IFN-gamma-induced member of the GBP family of GTPases isolated from macrophages.

We have cloned a new member of the interferon (IFN)-induced guanylate-binding protein (GBP) family of GTPases, murine GBP-2 (mGBP-2), from bone marrow-derived macrophages. mGBP-2 is located on murine chromosome 3, where it is linked to mGBP-1. With the identification of mGBP-2 there are now two human and two murine GBPs. Like other GBPs, mGBP-2 RNA and protein are induced by IFN-gamma. In addition, mGBP-2 shares with the other GBPs important structural features that distinguish this family from other GTPases. First, mGBP-2 contains only two of the three consensus sequences for nucleotide binding found within the classic GTP binding regions of other GTPases. A second amino acid motif found in mGBP-2 is a potential C-terminal site for isoprenoid modification, called a CaaX sequence. mGBP-2 is prenylated, as detected by [3H]mevalonate incorporation, when expressed in COS cells and preferentially incorporates the C-20 isoprenoid geranylgeraniol. Surprisingly, despite having a functional CaaX sequence, mGBP-2 is primarily cytosolic. GBP proteins are very abundant in IFN-exposed cells, but little is known about their function. mGBP-2 is expressed by IFN-gamma-treated cells from C57Bl/6 mice, whereas mGBP-1 is not. Thus, the identification of mGBP-2 makes possible the study of GBP function in the absence of a second family member.

Selective chemokine mRNA accumulation in the rat spinal cord after contusion injury.

Following traumatic injury to the spinal cord, hematogenous inflammatory cells including neutrophils, monocytes, and lymphocytes infiltrate the lesion in a distinct temporal sequence. To examine potential mechanisms for their recruitment, we measured chemokine mRNAs in the contused rat spinal cord, using specific and sensitive reverse transcriptase polymerase chain reaction (RT-PCR) dot-blot hybridization assays. The neutrophil chemoattractant GRO-alpha was 30-fold higher than control values at 6 hr postinjury and decayed rapidly thereafter. LIX, a highly related alpha-chemokine, also was elevated early postinjury. Monocyte chemoattractant peptide (MCP)-1 and MCP-5 mRNAs, potent chemoattractants for monocytes, were significantly elevated at the lesion epicenter at 12 and 24 hr postinjury and declined thereafter. Interferon-gamma-inducible protein, 10 kDa (IP-10), chemoattractant towards activated T-lymphocytes, was significantly elevated at 6 and 12 hr postinjury. The dendritic cell chemoattractant MIP-3alpha also was increased, perhaps contributing to the development of T-cell autoreactivity to neural components after spinal cord injury (SCI) in rats. Other beta-chemokines, including MIP-1alpha and RANTES (regulated on expression normal T-cell expressed and secreted), were minimally affected by SCI. Expression of chemokines, therefore, directly precedes the influx of target neutrophils, monocytes, and T-cells into the spinal cord postinjury, as noted previously. Thus, selective chemokine expression may be integral to inflammatory processes within the injured spinal cord as a mechanism of recruitment for circulating leukocytes.

Rat p67 GBP is induced by interferon-gamma and isoprenoid-modified in macrophages.

The guanylate binding proteins, GBPs, are a family of interferon-induced GTP-binding proteins that include the rat p67. We report here that rat p67, for which interferon regulation had not previously been demonstrated, is induced by IFN-gamma and also by LPS in both cultured bone marrow-derived macrophages and microglia. The basal level of rat p67 in macrophages is low but increases dramatically between 2 and 4 hours after treating cells with either IFN-gamma or LPS. It then remains elevated over the next 24 hours. Rat p67 is isoprenoid modified. The isoprenoid modification was detected in p67 isolated both from primary IFN-gamma-activated macrophages and when the gene for p67 was transfected into COS cells. This is the first demonstration of in vivo prenylation of a GBP. The interferon regulation and prenylation of rat p67 point toward this protein being significant in the functions of both activated macrophages and microglia.

Molecular cloning and functional characterization of human lymphotactin.

We describe the isolation of a cDNA that encodes human lymphotactin (Ltn), a new class of lymphocyte-specific chemokine. Human Ltn shows similarity to some members of the C-C chemokine family but has lost the first and third cysteine residues that are characteristic of the C-C and C-X-C chemokines. Ltn is chemotactic for lymphocytes but not for monocytes, a characteristic that makes it unique among chemokines. In addition, calcium flux desensitization studies indicate that Ltn uses a unique receptor. The human Ltn gene maps to a different chromosome than do the C-C and C-X-C chemokine families. Taken together, these characteristics indicate that Ltn is the first example of a new class of human chemokines with preferential effects on lymphocytes.

Cytokine production profile of early thymocytes and the characterization of a new class of chemokine.

By using a variety of molecular techniques, we have characterized the cytokine profile of pro-T cells. During this characterization we cloned a novel cytokine that has chemotactic activities. We have designated this cytokine lymphotactin. The structural analysis, chromosomal location, and biological properties of lymphotactin provide strong evidence that this cytokine represents a new class of chemokine.

Lymphotactin: a cytokine that represents a new class of chemokine.

In this study, the cytokine-producing profile of progenitor T cells (pro-T cells) was determined. During screening of a complementary DNA library generated from activated mouse pro-T cells, a cytokine designated lymphotactin was discovered. Lymphotactin is similar to members of both the Cys-Cys and Cys-X-Cys chemokine families but lacks two of the four cysteine residues that are characteristic of the chemokines. Lymphotactin is also expressed in activated CD8+ T cells and CD4-CD8- T cell receptor alpha beta + thymocytes. It has chemotactic activity for lymphocytes but not for monocytes or neutrophils. The gene encoding lymphotactin maps to chromosome one. Taken together, these observations suggest that lymphotactin represents a novel addition to the chemokine superfamily.

The murine immune response to the male-specific antigen mouse testicular cytochrome c.

Male and female A/J mice were examined for their ability to elicit T lymphocyte and antibody (Ab) responses to the male-specific Ag, mouse testicular cytochrome c (Mt cyt). T lymphocytes from both male and female mice primed in vivo responded to the Ag in in vitro proliferation assays, and the dose-response curves were statistically indistinguishable. In addition, similar levels of Ab to Mt cyt were observed in immunized male and female mice. The B cells producing the Ab had switched isotypes to IgG1 and IgG2a, indicating that the self-reactive T helper (Th) cells in male mice were functional. Thus, male mice do not appear to be immunologically tolerant to Mt cyt, at least at the Th and B lymphocyte levels. No evidence for disease was found in male mice primed with Mt cyt. Major histocompatibility complex (MHC) class II-positive antigen-presenting cells are present in the testes and these were shown in vitro to process and present Mt cyt to a T cell hybridoma specific for the synthetic peptide Mt cyt 93-104. However, the hybridoma was not activated in the absence of exogenous Mt cyt 93-104 or Mt cyt, indicating that endogenous Mt cyt is not normally processed in sufficient quantity to effectively load MHC class II molecules with this particular Mt cyt-derived peptide. Notwithstanding any immunologic privilege of the testes, the lack of tolerance to Mt cyt and its failure to elicit an autoimmune disease could extend from the low levels of processed Mt cyt Ag available for T cell recognition. The T cell response elicited by Mt cyt contrasts the lack of response to mouse somatic cytochrome c which differs from Mt cyt at 13 amino acid residues and is expressed in most tissues and at higher levels.

A single amino acid substitution in a cytochrome c T cell stimulatory peptide changes the MHC restriction element from one isotype (I-Ak) to another (I-Ek).

The binding sites of class II major histocompatibility complex (MHC) molecules can accommodate many seemingly diverse peptides. In the case of mouse class II molecules, it appears that in general, the I-A and I-E isotypes associate with different peptides. In this study we report an example where a single amino acid substitution in an I-Ak restricted peptide changes the restriction element to I-Ek. A T cell hybridoma, F6.A10, specific for the peptide 93-104 from mouse testicular cytochrome c (Mt cyt 93-104) was found to be restricted by I-Ak using class II molecule specific blocking monoclonal antibodies (mAb). The activation of this hybridoma by Mt cyt 93-104 was competitively inhibited by other peptides that bind to the I-Ak molecule but not by the peptide Mt cyt 93-104(A96) in which lysine at position 96 was substituted by alanine. This single amino acid substitution resulted in the ability of Mt cyt 93-104(A96) to activate the pigeon cytochrome c specific, I-Ek restricted, T cell hybridoma 2B4.11. The activation of 2B4.11 by Mt cyt 93-104(A96) was inhibited by peptides which bind to the I-Ek molecule but not by Mt cyt 93-104 and by mAb specific for I-Ek but not by mAb specific for I-Ak. These results suggest that the amino acid at position 96 may be an important anchor residue for both I-Ak and I-Ek binding but that peptides with different amino acid side chains are accommodated at that position by one or the other MHC class II isotype. Thus, in this particular case a single amino acid residue in the peptides determines the MHC class II isotype specificity.