Elsevier

Chemico-Biological Interactions

Volume 244, 25 January 2016, Pages 9-15
Chemico-Biological Interactions

Aluminum trichloride inhibits osteoblast mineralization via TGF-β1/Smad signaling pathway

https://doi.org/10.1016/j.cbi.2015.11.027Get rights and content

Highlights

  • AlCl3 decreased osteoblast mineralized matrix nodules in vitro.

  • AlCl3 inhibited mRNA and protein expressions for components of TGF-β1/Smad pathway.

  • AlCl3 increased mRNA expression of Smad7 which is an inhibitor of TGF-β1/Smad pathway.

Abstract

Osteoporosis is a major global public health problem. Aluminum (Al) exposure inhibits osteoblast mineralization and induces osteoporosis. However, the exact mechanism is not fully understood. The transforming growth factor β1 (TGF-β1)/Smad pathway is a major signaling cascade in regulating osteoblast mineralization. To investigate whether TGF-β1/Smad signaling pathway was involved in the Al-induced inhibition of osteoblast mineralization, osteoblasts were cultured and exposed to different concentrations of aluminum trichloride (AlCl3) (containing 0, 0.01, 0.02 and 0.04 mg/mL Al3+) for 24 h. We found that mineralized matrix nodules, mRNA expressions of alkaline phosphatase (ALP), type I collagen (Col I), TGF-β1, TGF-β type I receptor, TGF-β type II receptor and Smad4, protein expressions of TGF-β1 and p-Smad2/3, Smad2/3/4 trimeric complex were all decreased, whereas the mRNA expressions of Smad7 were increased in the AlCl3-treated groups compared with those in control. In conclusion, these results indicated that AlCl3 inhibited osteoblast mineralization via TGF-β1/Smad signaling pathway in rat osteoblasts. Our findings could provide novel insights into the mechanisms of action of AlCl3 in osteoporosis.

Introduction

Osteoporosis is a major global public health problem of older individuals [1], [2], [3]. In China, approximately 23% of the population over 60 years old, and 50% of the population over 80 years old, have been diagnosed with osteoporosis [4], [5]. Aluminum (Al) accumulation of bone increased with age is one of the important pathogenies for osteoporosis [6], [7], [8]. Osteoporosis is often attributed to the aberrant bone mineralization. Al inhibits osteoblast mineralization process, thereby impairing bone formation and resulting in osteoporosis [6], [9], [10], [11]. However, the inhibition mechanism of bone mineralization caused by Al remains elusive.

Transforming growth factor β1 (TGF-β1)/Smad signaling pathway plays a key role in regulating osteoblast mineralization [12], [13], [14], [15]. In TGF-β1/Smad signaling pathway, upon TGF-β1 ligand binding, the constitutively active type II receptor phosphorylates and type I receptor [16]. The activated type I receptor phosphorylates Smad2 and Smad3. In the following activation, Smad2 and Smad3 form a trimeric complex with Smad4, and this complex subsequently translocates into the nucleus and regulates downstream genes expression [16], [17]. Smad7 is an intracellular inhibitor, which is rapidly induced by TGF-β1 family members and provides a negative feedback loop [18]. TGF-β1/Smad signaling pathway regulates alkaline phosphatase (ALP) activity and the levels of type I collagen (Col I), which are markers for osteoblast mineralization [19]. However, it remains unknown whether TGF-β1/Smad signaling pathway is involved in the toxic effects of Al on osteoblast mineralization.

Therefore, the mineralized matrix nodules, the mRNA expressions of ALP, Col I, TGF-β1, TGF-β type I receptor, TGF-β type II receptor, Smad4 and Smad7, protein expressions of TGF-β1 and p-Smad2/3, and Smad2/3/4 trimeric complex in rat osteoblasts were examined to explore the effects of aluminum trichloride (AlCl3) on the TGF-β1/Smad signaling pathway and osteoblastic mineralization in rat osteoblasts cultured in vitro.

Section snippets

Cell culture and AlCl3 treatment

This experimental designs and procedures were approved by the Animal Ethics Committee of the Northeast Agricultural University (Harbin, China). Primary osteoblasts were isolated from the calvarium of 3-day-old Sprague-Dawley rats as previously described [10]. In brief, the calvariums were dissected aseptically and digested with 0.25% trypsin solution (Gibco, Grand Island, New York, USA) for 15 min at 37 °C, then in 0.1% collagenase type Ⅱ solution (Gibco, Grand Island, New York, USA) for 1 h.

Effects of AlCl3 on the formation of mineralized matrix nodules

To investigate the effect of AlCl3 on osteoblast mineralization, osteoblasts exposed to different concentrations of AlCl3 (containing 0, 0.01, 0.02 and 0.04 mg/mL Al3+) for 24 h. Alizarin red staining showed that with the increase of AlCl3 dosages, the number of mineralized matrix nodules were decreased, and were significantly lower in LG, MG and HG than those in CG (P < 0.01) (Fig. 1A and B). These results suggested that AlCl3 inhibited osteoblast mineralization in vitro.

The mRNA expressions of ALP, Col I, TGF-β1, TGF-β type I receptor, TGF-β type II receptor, Smad4 and Smad7

To investigate the

Discussion

The present study found that AlCl3 inhibited the osteoblast mineralization by decreased mineralized matrix nodules, mRNA expressions of ALP and Col I. Moreover, AlCl3 inhibited TGF-β1/Smad signaling pathway, presenting as the decreased mRNA expressions of TGF-β1, TGF-β type I receptor, TGF-β type II receptor and Smad4, protein expressions of TGF-β1 and p-Smad2/3, Smad2/3/4 complex, and increased Smad7 mRNA expression. The results proved that AlCl3 caused osteoblast mineralization dysfunction by

Conclusion

AlCl3 exposure inhibited mineralized matrix nodules formation, decreased gene expressions of ALP and Col I, and downregulated the TGF-β1/Smad signaling pathway. These results indicated that AlCl3 inhibits osteoblast mineralization via TGF-β1/Smad signaling pathway. Our findings provide novel insights into the mechanisms of action of AlCl3 in osteoporosis.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This study was supported by a grant from the Science and Technology Program of Heilongjiang Educational Bureau (C201425).

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