Differential effects of homocysteine and beta aminopropionitrile on preosteoblastic MC3T3-E1 cells Roman Thaler a , Silvia Spitzer a , Monika Rumpler a , Nadja Fratzl-Zelman a , Klaus Klaushofer a , Eleftherios P. Paschalis a,b , Franz Varga a, ⁎ a Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of WGKK and AUVA Trauma Center Meidling, 4th Medical Department, Hanusch Hospital, Vienna, Austria b University General Hospital “Attikon”, National Kapodistrian University of Athens, School of Medicine, 1st Department of Orthopaedic Surgery, Greece abstract article info Article history: Received 22 September 2009 Revised 26 October 2009 Accepted 27 October 2009 Available online 4 November 2009 Edited by: R. Baron Keywords: Osteoblast Collagen cross-links Gene expression Beta aminopropionitrile Homocysteine Compounds, like beta-aminopropionitrile (bAPN) and homocysteine (hcys), are known to inhibit a stable matrix formation. Osteoblast-synthesized collagen matrix regulates the differentiation of precursor cells into mature osteoblasts. They express lysyl oxidase, an enzyme involved in the collagen cross-linking process. Lately, plasma hcys levels have recently been strongly correlated with fracture in humans. We have previously shown that bAPN not only disturbs collagen cross-links but also affects osteoblastic differentiation in a cell culture system. The aim of the present study was to investigate the effects of bAPN and hcys on collagen cross-links and gene expression at the mRNA level by FTIR and quantitative RT-PCR, respectively. We found that bAPN and hcys down-regulated cell multiplication. While bAPN also down-regulated the metabolic activity of MC3T3-E1 cells, hcys down-regulated it by lower concentrations but up-regulated it by higher; both substances up- regulated alkaline phosphatase activity. The substances increased the ratio of pyr/divalent cross-links of collagen, and down-regulated mRNA expression of lysyl hydroxylase (Plod2) and lysyl oxidase (Lox), genes which play an important role in the formation of a stable matrix. Furthermore, we demonstrate that both substances stimulated the expression of Runx2, an indispensable regulator of osteoblastic differentiation. However, analysis of genome wide mRNA expression suggests that hcys and bAPN have differential effects on genes involved in osteoblastic differentiation and phenotype regulation. The results indicate that although both bAPN and hcys affect collagen cross-link post-translational modifications in a similar manner as far as pyr and divalent cross-links are concerned, they have differential effects on the monitored genes expression at the mRNA level, with hcys exerting a broader effect on the genome wide mRNA expression. © 2009 Elsevier Inc. All rights reserved. Introduction We have previously reported that beta-aminopropionitrile (bAPN), a known lathyrogen, not only disrupts collagen cross-linking but also affects osteoblastic activity and gene expression [1]. Homocysteine (hcys), a natural eukaryotic metabolite related to several chronic pathologies such as cardio-vascular diseases has also been shown to interfere with lysyl oxidase (Lox) action [2], altering collagen post-translational modifications and cross-links. However, an ambivalent classification of hcys as lathyrogen is found in literature. We have recently reported that there is a correlation between plasma hcys levels and collagen cross-link ratio in forming trabecular surfaces in humans [3]. Furthermore, recent clinical and epidemiological data [4–7] report a correlation between blood hcys levels and fracture risk. Clinical reports to the contrary also exist, as well as ones casting doubt as to whether hcys affects collagen cross- linking [8–10]. The situation does not become clearer when animal models are considered [11–15]. In the present study, using the preosteoblastic MC3T3-E1 cell line, the effects of hcys and bAPN on cell viability, cross-link formation and mRNA expression were compared as a function of concentration. Using quantitative reserve transcription polymerase chain reaction (qRT-PCR) and gene chip analysis, we investigated on the effects of both substances on the expression of genes involved in extracellular matrix formation, on general mRNA expression and on differentiation factors. Concerning cell viability and multiplication as well as expression of genes involved in post-translational collagen cross-linking, both interventions affected collagen in a similar manner. However, gene expression of selected gene groups res- ponsible for the osteoblastic phenotype and development was mostly differentially regulated by the used interventions. Further- more, a general gene array analysis revealed that hcys regulated at all concentrations more genes than bAPN and that the effects of hcys were generally stronger. Bone 46 (2010) 703–709 ⁎ Corresponding author. Fax: +43 1 91021 86929. E-mail address: franz.varga@osteologie.at (F. Varga). 8756-3282/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2009.10.038 Contents lists available at ScienceDirect Bone journal homepage: www.elsevier.com/locate/bone