Alendronate Inhibits PTH (1–34)-induced Bone Morphogenetic Protein Expression in MC3T3-E1 Preosteoblastic Cells

Abstract

The bisphosphonate class of antiresorptive drugs and active forms of parathyroid hormone (PTH (1–34)) have been used clinically to enhance bone mass and density in patients with osteoporosis. Abundant evidence suggests that the mechanism by which PTH (1–34) increases bone density is stimulation of osteoblast differentiation. Although bisphosphonates have been classically thought to increase bone density by inhibiting osteoclasts, there is increasing evidence to suggest that bisphosphonates have direct stimulatory effects on osteoblast differentiation. Interestingly, in patients with osteoporosis, combination therapy with bisphosphonates and PTH (1–34) is not synergistic in increasing bone density; bisphosphonates appear to blunt the effect of PTH (1–34). To begin to understand the mechanism governing the effects of these agents on osteoblasts and a possible explanation for their apparent antagonism, we examined the expression of several bone morphogenetic proteins (BMPs) in MC3T3-E1 preosteoblastic cells either untreated, or treated with alendronate, parathyroid hormone, or a combination of the two agents. We find by reverse transcriptase-polymerase chain reaction (RT-PCR) that while alendronate fails to induce the expression of any of the BMPs tested, several BMPs are induced by PTH (1–34). The induction of the PTH (1–34)-inducible BMPs is blocked with simultaneous alendronate treatment. These data suggest that alendronate interferes with PTH (1–34)-induced BMP gene transcription and provides a possible basis for the antagonism observed between the two agents in increasing bone density.

Introduction

Bone remodeling involves both resorption and bone formation—coupled processes that occur throughout life. When this highly regulated linkage is disrupted, the structural and material properties of bone diminish. Several human diseases, including postmenopausal osteoporosis, Paget’s disease, and fibrous dysplasia result from this uncoupling of bone formation and bone resorption. Low bone mineral density with increased risk of fragility and pathologic fractures results when the rate of resorption exceeds the rate of formation [1, 2].

Bisphosphonate drugs such as alendronate have been used to restore the balance between bone resorption and bone formation [1, 2]. There is abundant evidence suggesting that bisphosphonates act to decrease bone resorption by inhibiting the function of osteoclasts [1]. More recently, there has been increasing evidence to suggest that bisphosphonates may act to stimulate osteoblast differentiation [3–7]. Whereas the mechanism governing bisphosphonate-mediated osteogenesis is poorly understood, the evidence that alendronate can increase the expression of osteoblast-specific marker genes such as osteocalcin and type I collagen suggests that this regulation may be, in part, at the transcriptional level [7].

Parathyroid hormone (PTH) is an anabolic agent that enhances osteoblastic bone formation. Teriparatide, the active N-terminal fragment of PTH (residues 1–34), has been approved by the US Food and Drug Administration for the treatment of postmenopausal women with osteoporosis who are at high risk for fracture [2]. Because bisphosphonates have been shown to inhibit osteoclasts, a mechanism distinct from the anabolic effect of PTH (1–34) on osteoblasts, it has been suggested that their effects in osteoporosis may be additive. However, two clinical trials of the active form of PTH (1–34) and alendronate in men and postmenopausal women demonstrated that alendronate appeared to impair the ability of the active form of PTH (1–34) to increase bone mineral density [8–10]. The mechanism governing the antagonistic effects of combination therapy is unclear.

Bone morphogenetic proteins, including BMP-2, BMP-4, and BMP-7, play critical roles in osteoblast differentiation and bone formation and are thus good candidates for mediating the osteogenic signals of bisphosphonates and PTH (1–34) [11]. We therefore examined the expression of several bone morphogenetic proteins in osteoblasts either untreated or treated with alendronate, PTH (1–34), or a combination of the two agents. We are using the MC3T3-E1 preosteoblastic cell line, which has been widely used as a cell culture model for osteoblast differentiation. In response to several osteogenic signals such as the bone morphogenetic proteins or ascorbic acid, these cells express osteoblast-specific genes such as osteocalcin, core-binding factor alpha subunit 1 (Cbfa1), bone sialoprotein II, and type I collagen and undergo morphologic changes including mineralization [12–17]. We find by RT-PCR that BMP-1, which is not a member of the BMP/TGF-β family, and BMP-4 are constitutively expressed in untreated MC3T3-E1 cells, and treatment with alendronate does not alter this expression pattern. Treatment of MC3T3-E1 cells with PTH (1–34) induces expression of several of the BMPs; this induction is blocked by simultaneous alendronate treatment. Understanding the interaction of differentiation signals initiated by alendronate and PTH (1–34) will help provide a molecular basis for the design of better combination therapies to more effectively treat disorders of bone insufficiency.

Materials and methods

Materials and cell culture

PTH (1–34) (Forteo) containing the biologically active N-terminal 34 residues of parathyroid hormone was obtained from Eli Lilly (Indianapolis, IN, USA). Alendronate (Fosamax) was obtained from Merck and Co.(Whitehouse Station, NJ, USA). MC3T3-E1 preosteoblastic cells were cultured at 37°C, 10% CO₂, in 60-mm dishes, in α-MEM (Invitrogen) supplemented with 10% fetal bovine serum (Sigma) and 50 μg/ml penicillin and streptomycin. Cells were treated with either alendronate (10⁻⁸), PTH (1–34) (10^-7), or both, 24 h after plating (at 80% confluence). There was no additional media change upon treatment. Cells were harvested for RNA isolation 24 h after treatment.

RNA isolation and RT-PCR

Total RNA was isolated using Trizol Reagent (Invitrogen). Reverse transcriptase-PCR (RT-PCR) was performed using a reverse transcription kit (ReactionReady first strand cDNA synthesis kit; Super Array, Frederick, MD, USA ) and a PCR kit consisting of primers for mouse BMP genes (mouse BMP gene family multigene-12 RT-PCR profiling kit, PM-040B; Super Array, Fredrick, MD, USA). RT-PCR was performed according to the manufacturer’s instructions. PCR was performed using a thermal cycler (Applied Biosystems, Foster City, CA, USA). Ten microliters of PCR product was analyzed on a 2.0% agarose gel.

Results

Alendronate inhibits mRNA expression of several BMPs in PTH (1–34) treated MC3T3-E1 osteoblasts

We examined expression of several BMPs by RT-PCR in MC3T3-E1 cells treated with either alendronate (10⁻⁸), PTH (1–34) (10⁻⁷), or both (Fig. 1A–D). Concentrations were chosen based on published dose-response studies [7, 18, 19]. We note that BMP-1 and BMP-4 are constitutively expressed in MC3T3E1 cells (Fig. 1A). Alendronate does not induce expression of any of the BMPs over untreated MC3T3E1 cells (Fig. 1B). PTH (1–34) induces several BMPs including BMP-2, 3, 5, 6, 7, 8A, 8B, 10, and 3B (Fig. 1C). Interestingly, combination treatment with PTH (1–34) and alendronate blocks the mRNA expression of all nine PTH (1–34)-inducible BMPs (Fig. 1D).

Fig. 1.

RT-PCR for expression of BMP family members (BMP-1, 2, 3, 4, 5, 6, 7, 8A, 8B, 10, 3B) and GAPDH (lanes 1–12, respectively) in MC3T3-E1 cells either untreated (A) or treated with alendronate [10⁻⁸], (B) PTH (1–34) [10^-7] (C), or alendronate [10⁻⁸] and PTH (1–34) [10⁻⁷] (D). PCR products were resolved by 2.0% agarose gel electrophoresis

Discussion

The ability of bisphosphonates to inhibit osteoclasts coupled with the stimulatory effects of PTH (1–34) on osteoblasts suggests that their effects in increasing bone mineral density may be additive. However, recent clinical trials have demonstrated that alendronate appeared to impair the ability of PTH (1–34) to increase bone mineral density [8–10]. We observe that alendronate blocks the ability of PTH (1–34) to induce the mRNA expression of several BMPs in the preosteoblastic MC3T3-E1 cell line. This suggests a mechanism for the observed clinical antagonism between alendronate and PTH (1–34) in increasing bone density.

The BMPs encode a highly conserved class of signaling factors, which can induce ectopic bone formation in vivo. They are expressed during limb development, endochondral ossification, and early fracture healing. In several animal studies, BMPs have demonstrated the ability to enhance spinal fusion, repair critical-size defects, and accelerate fracture union [20, 21]. Interestingly, we find that BMP-1, distinct from the TGF-β-like BMPs, is constitutively expressed. BMP-1 is a tolloid-like proteinase, which plays critical roles in the synthesis of extracellular matrix proteins including several types of collagens and proteoglycans [22]. BMP-1 activates latent TGF-beta by cleaving latent TGF-b-binding protein [23] and can modulate BMP-2 and BMP-4 signaling [24]. Constitutive expression of BMP-1 in this cell type may reflect the preosteoblastic genoyype of these cells and their potential to differentiate down the osteoblastic pathway.

Two BMPs have been widely studied for their ability to induce bone regeneration in the clinical situation: recombinant human BMP-2 (rhBMP-2) and recombinant human BMP-7 (OP-1 [osteogenic protein-1]). These factors induce bone formation and healing of bone when surgically introduced [20, 21, 25]. In clinical trials, rhBMP-2 and OP-1 have been shown to accelerate the healing of open tibial fractures and tibial nonunions, respectively [26, 27]. The induction of BMP-2 and BMP-7 by PTH (1–34) is consistent with the bone-forming capacity of these agents and may partly explain the inductive effect of PTH on osteoblast differentiation. The block of this induction by alendronate can, in part, account for the antagonism observed between bisphosphonates and PTH (1–34) in increasing bone formation.

While increased BMP expression in PTH-treated MC3T3-E1 cells is suggestive of increased osteogenic potential, expression of other bone markers must be examined. There is evidence that PTH stimulates osteogenesis and increases expression of the bone-specific gene osteocalcin by increasing activity of the transcription factor osteoblast-specific factor 1 (osf1) [19]. If PTH regulates this process by induction of BMPs, future studies would be required to examine the ability of BMP antagonists to block the ability of PTH to increase osteocalcin gene transcription; this would confirm the necessity of BMP to transduce the PTH-mediated osteogenic signal.

Conclusion

The mechanism by which alendronate suppresses PTH (1–34)-induced BMP expression is unclear. The lack of expression of mRNAs for PTH (1–34)-inducible BMPs in MC3T3 E1 cells when treated with both PTH (1–34) and alendronate suggests that alendronate represses expression of these BMPs at the transcriptional level. Future studies aimed at identifying regions in the BMP-2 and BMP-7 promoters repressed by simultaneous alendronate and PTH (1–34) treatment and comparing these regions to PTH (1–34)-inducible elements in these promoters may provide insight into the early PTH (1–34)-mediated signal pathways interrupted by alendronate. Understanding the interactions of these signaling pathways is a first step toward designing superior combination therapies for treating low bone density and preventing pathologic fractures.