Interleukin-8 stimulation of osteoclastogenesis and bone resorption is a mechanism for the increased osteolysis of metastatic bone disease.

Interleukin 8 (IL-8) is a member of the alpha chemokine family of cytokines originally identified as a neutrophil chemoattractant. Recently, we reported that elevated levels of IL-8, but not parathyroid hormone-related protein (PTHrP), correlated with increased bone metastasis in a population of human breast cancer cells. We hypothesized that IL-8 expression by breast cancer cells would either indirectly influence osteoclastogenesis via nearby stromal cells or directly influence osteoclast differentiation and activity. In the present study, we investigated the role of IL-8 in the process of osteoclast formation and bone resorption, which is associated with metastatic breast cancer. The addition of recombinant human (rh) IL-8 (10 ng/ml) to cultures of stromal osteoblastic cells stimulated both RANKL mRNA expression and protein production, with no effect on the expression of osteoprotegerin. In addition, rhIL-8 also directly stimulated the differentiation of human peripheral blood mononuclear cells into bone-resorbing osteoclasts. In these cultures, IL-8 was able to stimulate human osteoclast formation even in the presence of excess (200 ng/ml) RANK-Fc. The effect of IL-8 on osteoclasts and their progenitors was associated with the cell surface expression of the IL-8-specific receptor (CXCR1) on the cells. These results demonstrate a direct effect of IL-8 on osteoclast differentiation and activity. Together, these data implicate IL-8 in the osteolysis associated with metastatic breast cancer.

Immunohistochemical study of osteopontin expression during distraction osteogenesis in the rat.

Distraction osteogenesis (DO) is a limb-lengthening procedure that combines mechanical tension stress with fracture healing to provide a unique opportunity for detailed histological examination of bone formation. Osteopontin (OPN) is a multifunctional matricellular protein believed to play a key role in wound healing and cellular response to mechanical stress. We studied the expression of OPN during DO using standard immunohistochemical (IHC) staining techniques. In addition, we compared the expression of OPN to proliferation (PCNA-positive cells) in the DO gap. After 14 days of distraction in the rat, these stains revealed variations in OPN expression and its relationship to proliferation according to the cell type, tissue type, and mode of ossification examined. Fibroblast-like cells within the central fibrous area exhibited intermittent low levels of OPN, but no relationship was observed between OPN and proliferation. In areas of transchondral ossification, OPN expression was very high in the morphologically intermediate oval cells. During intramembranous ossification, osteoblasts appeared to exhibit a bimodal expression of OPN. Specifically, proliferating pre-osteoblasts expressed osteopontin, but OPN was not detected in the post-proliferative pre-osteoblasts/osteoblasts that border the new bone columns. Finally, intracellular OPN was detected in virtually all of the mature osteoblasts/osteocytes within the new bone columns, while detection of OPN in the matrix of the developing bone columns may increase with the maturity of the new bone. These results imply that the expression of OPN during DO may be more similar to that seen during embryogenesis than would be expected from other studies. Furthermore, the biphasic expression of OPN during intramembranous ossification may exemplify the protein's multi-functional role. Early expression may facilitate pre-osteoblastic proliferation and migration, while the latter downregulation may be necessary for hydroxyapatite crystal formation.

Physiological role of mGSTA4-4, a glutathione S-transferase metabolizing 4-hydroxynonenal: generation and analysis of mGsta4 null mouse.

The lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) is a strong electrophile that forms covalent adducts with proteins and, to a lesser extent, nucleic acids and phospholipids. The generation of 4-HNE appears to be an inevitable consequence of aerobic metabolism. The metabolism of 4-HNE is mainly, although not entirely, conjugative, and proceeds via Michael addition of glutathione to the double bond of 4-HNE. This reaction is catalyzed by specialized glutathione S-transferases (GSTs) exemplified by the murine mGSTA4-4. To study the (patho)physiological effects of 4-HNE in an intact organism, we disrupted the mGsta4 gene in the mouse. The resulting mGsta4 null mouse expressed no mGsta4 mRNA and no corresponding protein, had a reduced ability to conjugate 4-HNE, and had an increased steady-state level of this aldehyde in tissues. The residual conjugating activity for 4-HNE (23-64% depending on the tissue) is probably attributable to isoforms of glutathione S-transferases which have low catalytic efficiency for 4-HNE but are more abundant than mGSTA4-4, or are upregulated upon mGsta4 gene disruption. Mice homozygous for the disrupted mGsta4 allele were viable and appeared normal except for lower litter size, higher fat content in bones, and greater susceptibility to bacterial infection. The null mice had a significantly lower survival time than wild-type controls when chronically treated with relatively low doses of paraquat, a finding consistent with a role of mGSTA4-4 in the defense against oxidative stress. The mouse model should be useful for the study of degenerative conditions in which 4-HNE is postulated to be a contributing factor.