Siddharth R Vora

Boston University, Boston, Massachusetts, United States

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Publications (10)52.04 Total impact

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    ABSTRACT: 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 FRS2alpha 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 (125)I-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.
    Journal of Biological Chemistry 03/2010; 285(10):7384-93. DOI:10.1074/jbc.M109.033597 · 4.60 Impact Factor
  • S. VORA, P. TRACKMAN
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    ABSTRACT: Introduction: Lysyl oxidase is secreted as a 50 kDa pro-enzyme and is then cleaved to a 30 kDa mature enzyme (LOX) and an 18 kDa propeptide (LOX-PP). Preliminary data indicate that LOX-PP inhibits proliferation of osteoblasts and delays terminal mineralization. FGF-2 is a key mitogen for osteoblasts that expands the pool of cells during early stages of osteoblast development. Here we test effects of LOX-PP on osteoblast proliferation with specific emphasis on FGF-2 signaling. Methods: Effects of recombinant LOX-PP protein on normal osteoblast proliferation and differentiation were determined in vitro. Results: LOX-PP inhibits FGF-2-induced DNA synthesis up to 70% in a dose dependent manner. Western blot analyses show that LOX-PP inhibits FGF-2-induced ERK1/2 by up to 65% in a dose dependent manner. Additionally, LOX-PP inhibits FGF-2-induced FRS2α phosphorylation and strongly inhibits FGF-2 stimulated DNA synthesis (up to 90%) under conditions where HSPG sulfation is inhibited, pointing to a target of LOX-PP at or near the level of FGF receptor. Competitive ligand binding assays on osteoblast cell layers demonstrate concentration dependent inhibition of FGF-2 binding by LOX-PP. Additionally, in vitro binding assays with recombinant FGF-receptor protein revealed that LOX-PP inhibition is non-competitive. Moreover, LOX-PP has an inhibitory effect on terminal bone nodule formation in osteoblast cultures only when added early, during the proliferative stage of osteoblast differentiation. No inhibitory effects are seen if LOX-PP is added during later stages of osteoblast differentiation. Conclusions: These data demonstrate that LOX-PP inhibits osteoblast proliferation and point to FGF-2 as a target for LOX-PP actions. We propose a working model for the respective roles of LOX enzyme as well as LOX-PP in osteoblast phenotype development, where LOX-PP acts to inhibit proliferation, potentially allowing cells to exit from the cell cycle and progress to the next stages of differentiation. NIH R01DE14066
    AADR Annual Meeting 2010; 03/2010
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    ABSTRACT: Lysyl oxidase enzyme activity is critical for the biosynthesis of mature and functional collagens and elastin. In addition, lysyl oxidase has tumor suppressor activity that has been shown to depend on the propeptide region (LOX-PP) derived from pro-lysyl oxidase (Pro-LOX) and not on lysyl oxidase enzyme activity. Pro-LOX is secreted as a 50 kDa proenzyme and then undergoes biosynthetic proteolytic processing to active approximately 30 kDa LOX enzyme and LOX-PP. The present study reports the efficient recombinant expression and purification of rat LOX-PP. Moreover, using enzymatic deglycosylation and DTT derivatization combined with mass spectrometry technologies, it is shown for the first time that rLOX-PP and naturally occurring LOX-PP contain both N- and O-linked carbohydrates. Structure predictions furthermore suggest that LOX-PP is a mostly disordered protein, which was experimentally confirmed in circular dichroism studies. Due to its high isoelectric point and its disordered structure, we propose that LOX-PP can associate with extracellular and intracellular binding partners to affect its known biological activities as a tumor suppressor and inhibitor of cell proliferation.
    Biochemistry 03/2010; 49(13):2962-72. DOI:10.1021/bi902218p · 3.19 Impact Factor
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    ABSTRACT: The lysyl oxidase (LOX) gene reverted Ras transformation of NIH 3T3 fibroblasts and tumor formation by gastric cancer cells, which frequently carry mutant RAS genes. The secreted lysyl oxidase proenzyme is processed to a propeptide (LOX-PP) and a functional enzyme (LOX). Unexpectedly, the tumor suppressor activity mapped to the LOX-PP domain, which inhibited tumor formation and the invasive phenotype of NF639 breast cancer cells driven by human epidermal growth factor receptor-2/neu, which signals via Ras. A single-nucleotide polymorphism, G473A (rs1800449), resulting in an Arg158Gln substitution in a highly conserved region within LOX-PP, occurs with an average 473A allele carrier frequency of 24.6% in the HapMap database, but was present in many breast cancer cell lines examined. Here, we show that the Arg-to-Gln substitution profoundly impairs the ability of LOX-PP to inhibit the invasive phenotype and tumor formation of NF639 cells in a xenograft model. LOX-PP Gln displayed attenuated ability to oppose the effects of LOX, which promoted a more invasive phenotype. In a case-control study of African American women, a potential association of the Gln-encoding A allele was seen with increased risk of estrogen receptor (ER)-alpha-negative invasive breast cancer in African American women. Consistently, LOX gene expression was higher in ER-negative versus ER-positive primary breast cancers, and LOX-PP Gln was unable to inhibit invasion by ER-negative cell lines. Thus, these findings identify for the first time genetic polymorphism as a mechanism of impaired tumor suppressor function of LOX-PP and suggest that it may play an etiologic role in ER-negative breast cancer.
    Cancer Research 09/2009; 69(16):6685-93. DOI:10.1158/0008-5472.CAN-08-4818 · 9.28 Impact Factor
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    ABSTRACT: 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 approximately 30 kDa LOX enzyme and approximately 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 FRS2alpha 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.
    Oncogene 08/2009; 28(38):3390-400. DOI:10.1038/onc.2009.203 · 8.56 Impact Factor
  • Cancer Research 01/2009; 69(2 Supplement):4073. DOI:10.1158/0008-5472.SABCS-4073 · 9.28 Impact Factor
  • Siddharth R. Vora, Philip C. Trackman
    Matrix Biology 12/2008; 27:27-27. DOI:10.1016/j.matbio.2008.09.288 · 3.65 Impact Factor
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    ABSTRACT: The lysyl oxidase (LOX) gene encodes an enzyme (LOX) critical for extracellular matrix maturation. The LOX gene has also been shown to inhibit the transforming activity of Ras oncogene signaling. In particular, the pro-peptide domain (LOX-PP) released from the secreted precursor protein (Pro-LOX) was found to inhibit the transformed phenotype of breast, lung, and pancreatic cancer cells. However, the mechanisms of action of LOX-PP remained to be determined. Here, the ability of LOX-PP to attenuate the integrin signaling pathway, which leads to phosphorylation of focal adhesion kinase (FAK), and the activation of its downstream target p130Cas, was determined. In NF639 breast cancer cells driven by Her-2/neu, which signals via Ras, ectopic Pro-LOX and LOX-PP expression inhibited fibronectin-stimulated protein tyrosine phosphorylation. Importantly, phosphorylation of FAK on Tyr-397 and Tyr-576, and p130Cas were substantially reduced. The amount of endogenous p130Cas in the Triton X-100-insoluble protein fraction, and fibronectin-activated haptotaxis were decreased. Interestingly, expression of mature LOX enzyme enhanced fibronectin-stimulated integrin signaling. Of note, treatment with recombinant LOX-PP selectively reduced fibronectin-mediated haptotaxis of NF639, MDA-MB-231, and Hs578T breast cancer cells. Thus, evidence is provided that one mechanism of action of LOX-PP tumor suppression is to block fibronectin-stimulated signaling and cell migration.
    Journal of Biological Chemistry 12/2008; 284(3):1385-93. DOI:10.1074/jbc.M802612200 · 4.60 Impact Factor
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    ABSTRACT: Lysyl oxidase is required for the normal biosynthesis and maturation of collagen and elastin. It is expressed by vascular smooth muscle cells, and its increased expression has been previously found in atherosclerosis and in models of balloon angioplasty. The lysyl oxidase propeptide (LOX-PP) has more recently been found to have biological activity as a tumor suppressor, and it inhibits Erk1/2 Map kinase activation. We reasoned that LOX-PP may have functions in normal non-transformed cells. We, therefore, investigated its effects on smooth muscle cells, focusing on important biological processes mediated by Erk1/2-dependent signaling pathways including proliferation and matrix metalloproteinase-9 (MMP-9) expression. In addition, we investigated whether evidence for accumulation of LOX-PP could be found in vivo in a femoral artery injury model. Recombinant LOX-PP was expressed and purified, and was found to inhibit primary rat aorta smooth muscle cell proliferation and DNA synthesis by more than 50%. TNF-alpha-stimulated MMP-9 expression and Erk1/2 activation were both significantly inhibited by LOX-PP. Immunohistochemistry studies carried out with affinity purified anti-LOX-PP antibody showed that LOX-PP epitopes were expressed at elevated levels in vascular lesions of injured arteries. These novel data suggest that LOX-PP may provide a feedback control mechanism that serves to inhibit properties associated with the development of vascular pathology.
    Biochemical and Biophysical Research Communications 03/2008; 366(1):156-61. DOI:10.1016/j.bbrc.2007.11.116 · 2.28 Impact Factor
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    ABSTRACT: Studies have shown that systemic PTH treatment enhanced the rate of bone repair in rodent models. However, the mechanisms through which PTH affects bone repair have not been elucidated. In these studies we show that PTH primarily enhanced the earliest stages of endochondral bone repair by increasing chondrocyte recruitment and rate of differentiation. In coordination with these cellular events, we observed an increased level of canonical Wnt-signaling in PTH-treated bones at multiple time-points across the time-course of fracture repair, supporting the conclusion that PTH responses are at least in part mediated through Wnt signaling. Since FDA approval of PTH [PTH(1-34); Forteo] as a treatment for osteoporosis, there has been interest in its use in other musculoskeletal conditions. Fracture repair is one area in which PTH may have a significant clinical impact. Multiple animal studies have shown that systemic PTH treatment of healing fractures increased both callus volume and return of mechanical competence in models of fracture healing. Whereas the potential for PTH has been established, the mechanism(s) by which PTH produces these effects remain elusive. Closed femoral fractures were generated in 8-wk-old male C57Bl/6 mice followed by daily systemic injections of either saline (control) or 30 microg/kg PTH(1-34) for 14 days after fracture. Bones were harvested at days 2, 3, 5, 7, 10, 14, 21, and 28 after fracture and analyzed at the tissue level by radiography and histomorphometry and at the molecular and biochemical levels level by RNase protection assay (RPA), real-time PCR, and Western blot analysis. Quantitative muCT analysis showed that PTH treatment induced a larger callus cross-sectional area, length, and total volume compared with controls. Molecular analysis of the expression of extracellular matrix genes associated with chondrogenesis and osteogenesis showed that PTH treated fractures displayed a 3-fold greater increase in chondrogenesis relative to osteogenesis over the course of the repair process. In addition, chondrocyte hypertrophy occurred earlier in the PTH-treated callus tissues. Analysis of the expression of potential mediators of PTH actions showed that PTH treatment significantly induced the expression of Wnts 4, 5a, 5b, and 10b and increased levels of unphosphorylated, nuclear localized beta-catenin protein, a central feature of canonical Wnt signaling. These results showed that the PTH-mediated enhancement of fracture repair is primarily associated with an amplification of chondrocyte recruitment and maturation in the early fracture callus. Associated with these cellular effects, we observed an increase in canonical Wnt signaling supporting the conclusion that PTH effects on bone repair are mediated at least in part through the activation of Wnt-signaling pathways.
    Journal of Bone and Mineral Research 01/2008; 22(12):1903-12. DOI:10.1359/jbmr.070724 · 6.59 Impact Factor

Publication Stats

279 Citations
52.04 Total Impact Points

Institutions

  • 2008–2009
    • Boston University
      • Department of Periodontology and Oral Biology
      Boston, Massachusetts, United States
    • University of Massachusetts Boston
      Boston, Massachusetts, United States
    • Beverly Hospital, Boston MA
      Beverly, Massachusetts, United States