Amitha H. Palamakumbura

The Tumor Suppressor Activity of the Lysyl Oxidase Propeptide Reverses the Invasive Phenotype of Her-2/neu–Driven Breast Cancer

Expression of the lysyl oxidase gene (LOX) was found to inhibit the transforming activity of the ras oncogene in NIH 3T3 fibroblasts and was hence named the ras recision gene (rrg). Lysyl oxidase (LOX) is synthesized and secreted as a 50-kDa inactive proenzyme (Pro-LOX), which is processed by proteolytic cleavage to a functional 32-kDa enzyme and an 18-kDa propeptide (LOX-PP). Recently, the ras recision activity of the LOX gene in NIH 3T3 cells was mapped to its propeptide region. Here, we show for the first time that LOX-PP inhibits transformation of breast cancer cells driven by Her-2/neu, an upstream activator of Ras. LOX-PP expression in Her-2/neu-driven breast cancer cells in culture suppressed Akt, extracellular signal-regulated kinase, and nuclear factor-kappaB activation. Her-2/neu-induced epithelial to mesenchymal transition was reverted by LOX-PP, as judged by reduced levels of Snail and vimentin; up-regulation of E-cadherin, gamma-catenin, and estrogen receptor alpha; and decreased ability to migrate or to form branching colonies in Matrigel. Furthermore, LOX-PP inhibited Her-2/neu tumor formation in a nude mouse xenograft model. Thus, LOX-PP inhibits signaling cascades induced by Her-2/neu that promote a more invasive phenotype and may provide a novel avenue for treatment of Her-2/neu-driven breast carcinomas.

A role for lysyl oxidase regulation in the control of normal collagen deposition in differentiating osteoblast cultures

Differentiation of phenotypically normal osteoblast cultures leads to formation of a bone‐like extracellular matrix in vitro. Maximum collagen synthesis occurs early in the life of these cultures, whereas insoluble collagen deposition occurs later and is accompanied by a diminished rate of collagen synthesis. The mechanisms that control collagen deposition seem likely to include regulation of extracellular collagen biosynthetic enzymes, but expression patterns of these enzymes in differentiating osteoblasts has received little attention. The present study determined the regulation of lysyl oxidase as a function of differentiation of phenotypically normal murine MC3T3‐E1 cells at the level of RNA and protein expression and enzyme activity. In addition, the regulation of BMP‐1/mTLD mRNA levels that encodes procollagen C‐proteinases was assayed. The role of lysyl oxidase in controlling insoluble collagen accumulation was further investigated in inhibition studies utilizing β‐aminopropionitrile, a specific inhibitor of lysyl oxidase enzyme activity. Results indicate that lysyl oxidase is regulated as a function of differentiation of MC3T3‐E1 cells, and that the maximum increase in lysyl oxidase activity precedes the most efficient phase of insoluble collagen accumulation. By contrast BMP‐1/mTLD is more constitutively expressed. Inhibition of lysyl oxidase in these cultures increases the accumulation of abnormal collagen fibrils, as determined by solubility studies and by electron microscopy. Taken together, these data support that regulation of lysyl oxidase activity plays a key role in the control of collagen deposition by osteoblast cultures. Copyright

Lysyl oxidase propeptide inhibits prostate cancer cell growth by mechanisms that target FGF-2-cell binding and signaling.

Enhanced RAS signaling and decreased androgen dependence of prostate cancer cells accompany poor clinical outcomes. Elevated autocrine fibroblast growth factors 2 (FGF-2) signaling promotes prostate cancer cell growth and survival. Expression of lysyl oxidase (LOX) inhibits RAS transforming activity. LOX is secreted as 50 kDa pro-LOX protein and then undergoes extracellular proteolytic processing to form ∼30 kDa LOX enzyme and ∼18 kDa propeptide (LOX-PP). We have previously shown that LOX-PP inhibits breast cancer cell transformation and tumor formation, but mechanisms of action of LOX-PP have not been fully elucidated. Here we report that LOX expression is reduced in prostate cancer cell lines and that recombinant LOX-PP protein inhibits serum-stimulated DNA synthesis and MEK/ERK and PI3K/AKT pathways in DU 145 and PC-3 androgen-independent cell lines. In DU 145 cells, treatment with a pharmacologic FGF-receptor inhibitor or a neutralizing anti-FGFR1 antibody mimicked LOX-PP inhibition of serum-stimulated DNA synthesis. FGF-2-stimulated DNA synthesis, ERK1/2, AKT and FRS2α activation were found all to be inhibited by LOX-PP in DU 145 cells. LOX-PP reduced specific binding of FGF-2 to DU 145 cells, suggesting that LOX-PP targets FGF signaling at the receptor. Interestingly, PC-3 cells did not respond to FGF-2, consistent with previous reports. We conclude that LOX-PP inhibits proliferation of DU 145 cells by interfering with FGFR(s) binding and signaling, and that LOX-PP has other mechanisms of action in PC-3 cells.

Tissue-specific mechanisms for CCN2/CTGF persistence in fibrotic gingiva: interactions between cAMP and MAPK signaling pathways, and prostaglandin E2-EP3 receptor mediated activation of the c-JUN N-terminal kinase.

Prostaglandin E2 blocks transforming growth factor TGF β1-induced CCN2/CTGF expression in lung and kidney fibroblasts. PGE2 levels are high in gingival tissues yet CCN2/CTGF expression is elevated in fibrotic gingival overgrowth. Gingival fibroblast expression of CCN2/CTGF in the presence of PGE2 led us to compare the regulation of CCN2/CTGF expression in fibroblasts cultured from different tissues. Data demonstrate that the TGFβ1-induced expression of CCN2/CTGF in human lung and renal mesangial cells is inhibited by 10 nm PGE2, whereas human gingival fibroblasts are resistant. Ten nm PGE2 increases cAMP accumulation in lung but not gingival fibroblasts, which require 1 μm PGE2 to elevate cAMP. Micromolar PGE2 only slightly reduces the TGFβ1-stimulated CCN2/CTGF levels in gingival cells. EP2 prostaglandin receptor activation with butaprost blocks the TGFβ1-stimulated expression of CCN2/CTGF expression in lung, but not gingival, fibroblasts. In lung fibroblasts, inhibition of the TGFβ1-stimulated CCN2/CTGF by PGE2, butaprost, or forskolin is due to p38, ERK, and JNK MAP kinase inhibition that is cAMP-dependent. Inhibition of any two MAPKs completely blocks CCN2/CTGF expression stimulated by TGFβ1. These data mimic the inhibitory effects of 10 nm PGE2 and forskolin that were dependent on PKA activity. In gingival fibroblasts, the sole MAPK mediating the TGFβ1-stimulated CCN2/CTGF expression is JNK. Whereas forskolin reduces TGFβ1-stimulated expression of CCN2/CTGF by 35% and JNK activation in gingival fibroblasts, micromolar PGE2-stimulated JNK in gingival fibroblasts and opposes the inhibitory effects of cAMP on CCN2/CTGF expression. Stimulation of the EP3 receptor with sulprostone results in a robust increase in JNK activation in these cells. Taken together, data identify two mechanisms by which TGFβ1-stimulated CCN2/CTGF levels in human gingival fibroblasts resist down-regulation by PGE2: (i) cAMP cross-talk with MAPK pathways is limited in gingival fibroblasts; (ii) PGE2 activation of the EP3 prostanoid receptor stimulates the activation of JNK.

Lysyl Oxidase Propeptide Inhibits FGF-2-induced Signaling and Proliferation of Osteoblasts

Pro-lysyl oxidase is secreted as a 50-kDa proenzyme and is then cleaved to a 30-kDa mature enzyme (lysyl oxidase (LOX)) and an 18-kDa propeptide (lysyl oxidase propeptide (LOX-PP)). The presence of LOX-PP in the cell layers of phenotypically normal osteoblast cultures led us to investigate the effects of LOX-PP on osteoblast differentiation. Data indicate that LOX-PP inhibits terminal mineralization in primary calvaria osteoblast cultures when added at early stages of differentiation, with no effects seen when present at later stages. LOX-PP was found to inhibit serum- and FGF-2-stimulated DNA synthesis and FGF-2-stimulated cell growth. Enzyme-linked immunosorbent assay and Western blot analyses show that LOX-PP inhibits FGF-2-induced ERK1/2 phosphorylation, signaling events that mediate the FGF-2-induced proliferative response. LOX-PP inhibits FGF-2-stimulated phosphorylation of FRS2α and FGF-2-stimulated DNA synthesis, even after inhibition of sulfation of heparan sulfate proteoglycans. These data point to a LOX-PP target at or near the level of fibroblast growth factor receptor binding or activation. Ligand binding assays on osteoblast cell layers with 125I-FGF-2 demonstrate a concentration-dependent inhibition of FGF-2 binding to osteoblasts by LOX-PP. In vitro binding assays with recombinant fibroblast growth factor receptor protein revealed that LOX-PP inhibits FGF-2 binding in an uncompetitive manner. We propose a working model for the respective roles of LOX enzyme and LOX-PP in osteoblast phenotype development in which LOX-PP may act to inhibit the proliferative response possibly to allow cells to exit from the cell cycle and progress to the next stages of differentiation.

A procollagen C-proteinase inhibitor diminishes collagen and lysyl oxidase processing but not collagen cross-linking in osteoblastic cultures.

The deposition of insoluble functional collagen occurs following extracellular proteolytic processing of procollagens by procollagen N‐ and C‐proteinases, fibril formation, and lysyl oxidase dependent cross‐linking. Procollagen C‐proteinases in addition process and activate lysyl oxidase. The present study evaluates a possible role for procollagen C‐proteinases in controlling different aspects of collagen deposition in vitro. Studies determine whether inhibition of procollagen C‐proteinase activity with a specific BMP‐1 inhibitor results in perturbations in lysyl oxidase activation, and in collagen processing, deposition, and cross‐linking in phenotypically normal cultured murine MC3T3‐E1 cells. Data show that BMP‐1 Inhibitor dose dependently inhibits lysyl oxidase activation by up to 50% in undifferentiated proliferating cells. In differentiating cultures, BMP‐1 inhibitor decreased collagen processing but did not inhibit the accumulation of mature collagen cross‐links. Finally, electron microscopy studies show that collagen fibril diameter increased. Thus, inhibition of procollagen C‐proteinases results in perturbed collagen deposition primarily via decreased collagen processing.