Intermittent glucocorticoid steroid dosing enhances muscle repair without eliciting muscle atrophy.

Glucocorticoid steroids such as prednisone are prescribed for chronic muscle conditions such as Duchenne muscular dystrophy, where their use is associated with prolonged ambulation. The positive effects of chronic steroid treatment in muscular dystrophy are paradoxical because these steroids are also known to trigger muscle atrophy. Chronic steroid use usually involves once-daily dosing, although weekly dosing in children has been suggested for its reduced side effects on behavior. In this work, we tested steroid dosing in mice and found that a single pulse of glucocorticoid steroids improved sarcolemmal repair through increased expression of annexins A1 and A6, which mediate myofiber repair. This increased expression was dependent on glucocorticoid response elements upstream of annexins and was reinforced by the expression of forkhead box O1 (FOXO1). We compared weekly versus daily steroid treatment in mouse models of acute muscle injury and in muscular dystrophy and determined that both regimens provided comparable benefits in terms of annexin gene expression and muscle repair. However, daily dosing activated atrophic pathways, including F-box protein 32 (Fbxo32), which encodes atrogin-1. Conversely, weekly steroid treatment in mdx mice improved muscle function and histopathology and concomitantly induced the ergogenic transcription factor Krüppel-like factor 15 (Klf15) while decreasing Fbxo32. These findings suggest that intermittent, rather than daily, glucocorticoid steroid regimen promotes sarcolemmal repair and muscle recovery from injury while limiting atrophic remodeling.

Two translation initiation codons direct the expression of annexin VI 64kDa and 68kDa isoforms.

Annexin A6 is a multicompetent, multifunctional protein involved in several biological processes within and outside of the cell. Whereas HeLa cells express annexin A6 only as a 68/67-kDa doublet, indicating alternative splicing (Smith PD et al. (1994) Proc Natl Acad Sci USA 91, 2713-2717), the GMO2784 human fibroblast cell line expresses two additional isoforms at 64 and 58kDa. In both cell lines, annexin A6 is located intracellularly and on the plasma membrane. In vitro eukaryotic protein synthesis of pIRESneoAnxA6 cDNA and pIRESneoAnxA6/Met1- or Met33- using a reticulocyte lysate coupled transcription/translation system revealed that this gene contains two translation start codons, Met1 and Met33. Immunoprecipitation of the products obtained from the transcription/translation system using various anti-annexin A6 antibodies confirmed the presence of several isoforms and suggested that this protein might be present in different configurations.

Identification of novel markers for monitoring minimal residual disease in acute lymphoblastic leukemia.

To identify new markers of minimal residual disease (MRD) in B-lineage acute lymphoblastic leukemia (ALL), gene expression of leukemic cells obtained from 4 patients with newly diagnosed ALL was compared with that of normal CD19(+)CD10(+) B-cell progenitors obtained from 2 healthy donors. By cDNA array analysis, 334 of 4132 genes studied were expressed 1.5- to 5.8-fold higher in leukemic cells relative to both normal samples; 238 of these genes were also overexpressed in the leukemic cell line RS4;11. Nine genes were selected among the 274 overexpressed in at least 2 leukemic samples, and expression of the encoded proteins was measured by flow cytometry. Two proteins (caldesmon and myeloid nuclear differentiation antigen) were only weakly expressed in leukemic cells despite strong hybridization signals in the array. By contrast, 7 proteins (CD58, creatine kinase B, ninjurin1, Ref1, calpastatin, HDJ-2, and annexin VI) were expressed in B-lineage ALL cells at higher levels than in normal CD19(+)CD10(+) B-cell progenitors (P <.05 in all comparisons). CD58 was chosen for further analysis because of its abundant and prevalent overexpression. An anti-CD58 antibody identified residual leukemic cells (0.01% to 1.13%; median, 0.03%) in 9 of 104 bone marrow samples from children with ALL in clinical remission. MRD estimates by CD58 staining correlated well with those of polymerase chain reaction amplification of immunoglobulin genes. These results indicate that studies of gene expression with cDNA arrays can aid the discovery of leukemia markers. (Blood. 2001;97:2115-2120)

Identification and categorization of inducible mast cell genes in a subtraction library.

Mast cells play an important role in allergic inflammation by releasing inducible proinflammatory cytokines. While many inducible genes have been identified, we hypothesized that a significant number remain to be identified. We thus constructed an activation-specific mast cell subtraction library to establish a profile of induced genes in mast cells following allergic stimulation. To date, we have sequenced 150 cDNA clones. Among them, we have isolated 22 known genes whose expression has not been reported in mast cells, and an additional 26 cDNA clones which do not have significant homology to known genes in the Genbank database. We next selected 10 cDNA clones with strong signals by differential plaque hybridization. Of these cDNA clones, five genes were induced in mast cells upon Fc epsilon RI-mediated stimulation. They are cofilin, annexinVI, interferon (IFN)-beta, serglycin, and a novel inducible mast cell (IMC) gene, IMC-415. Characterization and relevant studies of this novel gene and other inducible known genes in mast cells will provide insight into the functions of mast cells in mammalian biology.

cDNA cloning and tissue-specific regulation of expression of rat calcium-binding protein 65/67. Identification as a homologue of annexin VI.

We isolated a cDNA encoding the rat membrane-associated 65/67-kDa calcium-binding protein, CBP 65/67, from a lambda ZAP II cDNA-expression library of rat liver by immunoscreening using monospecific polyclonal anti-(CBP 65/67) antibodies and monoclonal anti-(CBP 65/67) IgG. The product of this cDNA expressed in Escherichia coli was confirmed as CBP 65/67 both by immunostaining and by comparison of the molecular mass with the CBP 65/67 isolated from rat liver by SDS/PAGE. The cDNA sequence and the deduced amino acid sequence of CBP 65/67 both show a high degree of identity to human p68 and human calelectrin, which belong to a family of calcium-dependent, membrane-associated, phospholipid-binding proteins, called annexins. This means that CBP 65/67 is a homolog of the two human proteins just mentioned above. We are not aware that a rat annexin VI has previously been isolated and sequenced. The mRNA expression of CBP 65/67 in different rat organs during development was investigated by Northern blot analysis. In adult tissues, high mRNA levels of CBP 65/67 were found in lung, heart, muscle, spleen and especially in thymus and pancreas, whereas in liver, kidney, intestine, stomach and brain only low levels of CBP 65/67 mRNA could be detected. The amount of mRNA during tissue development in kidney, stomach and muscle showed only slight changes. In contrast, a significant increase of CBP 65/67 expression was observed in liver, lung, heart and brain. In most of the organs investigated, the level of mRNA correlated closely with the level of protein expression, indicating that the expression of CBP 65/67 in most organs is controlled primarily at the transcriptional level.

Annexin VI isoforms are differentially expressed in mammalian tissues.

Purified annexin VI migrates as a closely spaced doublet when separated by SDS-PAGE. Immunolocalization of annexin VI in heart demonstrates staining at different defined subcellular compartments. Moss et al. identified two cDNAs, one having an insert of 18 bases encoding VAAEIL at the beginning of repeat domain seven. We have identified the splicing site of the murine annexin VI gene. It contains a single small exon of 18 bases. PCR amplification of reverse transcribed (RT) mRNA demonstrates that, in all tissues tested, the mRNA isoform containing the insert is predominant. Site-directed antibody was produced and affinity purified against peptides reflecting the insert and deletion sequences. The steady-state isoform ratio of the annexin VI protein is consistent with the RT-PCR data. Chromatographic experiments demonstrate that the annexin VI protein isoforms have biochemical differences. These differences may target the individual isoforms to unique cellular compartments or alter functional properties.