Velidi H. Rao

Expression and localization of 92 kDa type IV collagenase/gelatinase B (MMP-9) in human gliomas

Matrix metalloproteinases play an important regulatory role in tissue morphogenesis, cell differentiation and motility, and tumor cell invasiveness. We have recently demonstrated elevated activity of the 92 kDa type IV collagenase (MMP-9) in human glioblastoma and in the present study examine the relative amounts of MMP-9 protein and mRNA in human gliomas and as well as the distribution of MMP-9 in human glioma tumors in vivo. Using an enzyme-linked immunosorbent assay for the quantitative determination of MMP-9 protein, we found that levels were significantly higher in malignant astrocytomas, especially in glioblastoma multiforme, than in normal brain tissues and low-grade gliomas. In addition, the amount of MMP-9 mRNA, as determined by northern blot analysis was higher in anaplastic astrocytomas and glioblastoma multiforme than in normal brain tissue and low-grade gliomas. Immunocytochemical staining for MMP-9 showed strong cytoplasmic immunoreactivity in the tumor cells and the proliferating endothelial cells of glioblastoma multiforme and anaplastic astrocytomas. The staining intensity was lower in low-grade astrocytomas, and was undetectable or very low in normal brain astrocytes. The results indicate that expression of MMP-9 is dramatically upregulated in highly malignant gliomas and correlates with the highly malignant progression of human gliomas in vivo, and support a role for the MMP-9 in facilitating the invasiveness seen in malignant gliomas in vivo.

Expression of 72 kDa and 92 kDa type IV collagenases from human giant-cell tumor of bone.

Basement membrane forms widespread barriers to tumor invasion. It has been shown that tumor-secreted, basement membrane-degrading enzymes, namely metalloproteinases (MMPs) play an important role in tumor invasion and metastasis. In this study, we determined the enzymatic activity, content, and mRNA of both the 72 kDa (MMP-2) and 92 kDa (MMP-9) MMPs in primary cultures of human giant-cell tumor of bone (GCT)in vitro and in tissue extracts (in vivo). Gelatin zymography showed the presence of lytic bands at Mr 121000, 92000, and 72000, and these enzymatic activities were inhibited by EDTA, an inhibitor of MMPs. Western blots with antibodies specific for MMP-2 and MMP-9 confirmed the presence of MMP-2 and MMP-9 bothin vitro andin vivo, but GCT cells at late passage showed only MMP-2. Northern blots using labeled cDNA probes specific for these molecules revealed the presence of 3.1 kb transcript for MMP-2 and a 2.9 kb transcript for MMP-9. Using specific antibodies to 72 kDa and 92 kDa type IV collagenases, we studied their cellular distribution by immunohistochemical means. Stronger immunoreactivity was found for 92 kDa type IV collagenase than 72 kDa type IV collagenase in the giant cells. It appears, therefore, that MMP-9 may play an important role in the malignant behavior of GCTs and suggests a potential therapeutic role for protease inhibitors in attempting to minimize the invasive behavior of GCTs.

A cysteine for glycine substitution at position 175 in an α1 (i) chain of type I collagen produces a clinically heterogeneous form of osteogenesis imperfecta

The molecular basis for Osteogenesis Imperfecta in a large kindred with a highly variable phenotype was identified by sequencing the mutant pro alpha 1 (I) protein, cDNA and genomic DNA from the proband. Fibroblasts from different affected individuals all synthesize both normal Type I procollagen molecules and abnormal Type I procollagen molecules in which one or both pro alpha 1 (I) chain(s) contain a cysteine residue within the triple helical domain. Protein studies of the proband localized the mutant cysteine residue to the alpha 1 (I) CB 8 peptide. We now report that cysteine has replaced glycine at triple helical residue 175 disrupting the invariant Gly-X-Y structural motif required for perfect triple helix formation. The consequences include post-translational overmodification, decreased thermal stability, and delayed secretion of mutant molecules. The highly variable phenotype in the present kindred cannot be explained solely on the basis of the cysteine for glycine substitution but will require further exploration.

Interleukin-1β Upregulates MMP-9 Expression in Stromal Cells of Human Giant Cell Tumor of Bone

Giant cell tumor (GCT) of bone is a progressive, potentially malignant process that destroys skeletal tissue. It consists of multinucleated giant cells, which are hypothesized to be derived from a monocyte/macrophage lineage and mononuclear stromal cells, and the precise relationship of these cells is not fully understood. Recently, we demonstrated that the production of matrix metalloproteinase-9 (MMP-9) in GCT stromal cells is regulated by certain factor(s) secreted by the multinucleated giant cells. In the present study, we evaluated for the presence of interleukin-1β (IL-1β) and attempted to establish its possible role for the induction of MMP-9 in GCT stromal cells. Using enzyme-linked immunosorbent assay (ELISA), we have demonstrated that the primary GCT cultures secrete both IL-1β and MMP-9. The addition of monoclonal antibody (mAb) against IL-1β partially abrogated, but did not abolish, MMP-9 expression. Our results on gelatin zymography, reverse transcriptase-polymerase chain reaction (RT-PCR), a...

Normal Production, Nature, and Extent of Intracellular Degradation of Newly Synthesized Collagen in Fibroblasts from a Patient with Prolidase Deficiency

We have examined the extent of intracellular degradation of newly synthesized collagen occurring in fibroblasts from a patient with prolidase deficiency, a rare, autosomal recessively inherited disorder, in which a lack of prolidase, which normally cleaves imidodipeptides with a C-terminal Pro or Hyp residue, results in hyperimidodipeptiduria. The main clinical feature of the condition is chronic, intractable ulceration of the skin, and the suggestion has been made that it represents a specific disorder of collagen metabolism. Although most of the hydroxy-[14]proline derived from the intracellular degradation of newly synthesized collagen in prolidase-deficient fibroblasts occurred in imidodipeptides, with a similar chromatographic profile to those occurring in the patients urine, the proportion of collagen undergoing such degradation was as in control cells. No abnormality was found in other parameters of collagen metabolism studied, and the results confirm that, although the pathogenesis of its clinical manifestations remains unclear, the disorder is one of protein degradation in general.

Matrix metalloproteinases and their inhibitors in tumor invasion and metastasis

The recent progress in matrix metalloproteinases (MMPs) research has opened up many cellular targets in a variety of diseases. We have discussed some of the interesting and newly described roles of MMPs in modulating metastatic phenotype in malignancies. This information suggests a more specific and exclusive role of MMPs as important regulators of tumor cell invasion and metastasis. A great deal of room exists for redefining our current understanding of the MMPs system; the more we understand about how MMPs act, the better we will understand the pathobiology of metastasis. Research in the regulation of MMPs and the potential use of MMPs for therapeutic interventions in metastasis continues to expand. The future of therapy involving inhibitors of MMPs as a part of the armamentarium against human neoplasm should be viewed with cautious optimism. This therapeutic approach is beginning to find a place in our understanding of metastatic tumors, but with perhaps few exceptions should still be considered experimental. This area of research now needs more biological knowledge and imagination on the part of investigators to make future achievements possible.

Accelerated Linear Growth and Advanced Bone Age in Sotos Syndrome is Not Associated with Abnormalities of Collagen Metabolism

OBJECTIVES To investigate whether the advanced bone age in Sotos syndrome is associated with alterations in type I collagen metabolism in bone. DESIGN AND METHODS The metabolism of collagen was studied by analyzing the production, gene expression and degradation of type I collagen in dermal fibroblast strains from patients with Sotos syndrome and comparing them with fibroblasts from age-matched healthy subjects. Collagen production was determined as collagenase digestible radioactivity and collagen mRNA levels were measured by RT-PCR. Collagen degradation was assessed by specific collagenase assay and gelatin zymography. To determine the structural defects in type I collagen, the newly synthesized proteins were analyzed by SDS-PAGE before and after proteolytic digestion with pepsin. RESULTS In the present study, we have demonstrated that the collagen production, secretion and degradation in Sotos syndrome is comparable to controls. In addition, no qualitative differences in mRNA transcripts for type I collagen were detected between the control and Sotos syndrome fibroblasts. The secretion and intracellular accumulation of procollagen is also comparable to controls. The analysis of both procollagen and collagen on SDS-PAGE did not exhibit any major structural changes as compared with controls. CONCLUSIONS Our results on several aspects of collagen metabolism have demonstrated for the first time that collagen, the most abundant of mammalian proteins and the major constituent of bone, is normal in patients with Sotos syndrome. Therefore, it appears that the advanced bone age and accelerated linear growth seen in patients with Sotos syndrome may not be attributed to inherent abnormalities of collagen metabolism. The etiology and the pathogenesis of Sotos syndrome still remains unclear.