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. Author manuscript; available in PMC: 2009 Jun 2.
Published in final edited form as: J Steroid Biochem Mol Biol. 2006 Dec 22;103(3-5):606–609. doi: 10.1016/j.jsbmb.2006.12.083

Beta-1 Integrins Mediate Substrate Dependent Effects of 1α,25(OH)2D3 on Osteoblasts

Zvi Schwartz 1,2, Bryan F Bell 1, Liping Wang 3, Ge Zhao 1, Rene Olivares-Navarrete 1, Barbara D Boyan 1
PMCID: PMC2689367  NIHMSID: NIHMS20758  PMID: 17317155

Abstract

Surface microroughness increases osteoblast differentiation and enhances responses of osteoblasts to 1,25-dihydroxyvitamin D3 [1α,25(OH)2D3]. β1 integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and it is regulated by 1α,25(OH)2D3 in a surface-dependent manner, suggesting that it has a role in mediating osteoblast response. Here, we silenced β1 expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA) and examined the responses of the β1-silenced osteoblasts to surface microtopography and 1α,25(OH)2D3. MG63 cells were also treated with two different monoclonal antibodies to human β1 to block ligand binding. β1-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor β1, prostaglandin E2, and osteoprotegerin in comparison with control cells. Moreover, β1-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1α,25(OH)2D3. Anti β1 antibody AIIB2 had no significant effect on cell number and osteocalcin, but decreased alkaline phosphatase; MAB2253Z decreased cell number and alkaline phosphatase and increased osteocalcin in a dose-dependent manner. Effects of 1α,25(OH)2D3 on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that β1 plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1α,25(OH)2D3. The results also show that β1 mediates, in part, the synergistic effects of surface roughness and 1α,25(OH)2D3.

Keywords: 1α, 25(OH)2D3, β1 integrin subunit, Ti surface roughness, osteoblasts

INTRODUCTION

We and others have shown that osteoblasts are sensitive to micron scale and submicron scale structural properties of their substrate. These studies, recently reviewed by Bachle and Kohal [1], demonstrate that osteoblast proliferation is decreased on surfaces with rougher microtopographies whereas differentiation is enhanced. The in vitro observations are correlated with in vivo assessments of pull-out strength. There is greater bone-to-implant contact when implants with rough microtopographies are used and greater torque is required to remove the implants after bone healing [2–4].

The effects of surface microstructure on osteoblast behavior depend on the actual topography, including the shape of any spikes and the dimensions of any craters. We have used photolithography to fabricate titanium substrates with specific microfeatures and have found that osteoblast differentiation is greater when the cells are cultured in the presence of craters between 30 to 100 micrometers in diameter that are overlaid with a lawn of pits formed by spikes less than 1 micrometer in height. These conditions approximate those found on commercial Ti surfaces that have been sand blasted followed by an acid etch procedure (SLA). The SLA surface has an overall average roughness (Ra) of 4–5 micrometers. Roughness can also be achieved by forming a coating of irregular protrusions using a Ti plasma spraying technique (TPS), resulting in an Ra of 5–7 micrometers. These relatively small differences in surface shape can result in altered surface chemistry, not only because different methods are used for producing them but also because of differences in resulting surface energy. Thus, the response of an osteoblast to its substrate reflects a combination of factors including the physical structure, the chemical environment, and surface energy.

To study the regulation of osteoblast phenotype by surface structural elements, we have taken advantage of the SLA and TPS substrates provided to us by Institut Straumann AG (Basel, Switzerland), as well as of the smooth pretreatment substrate (PT) used as a base for SLA and TPS fabrication. Because these substrates are produced using the same manufacturing conditions used to produce dental implants, quality control is high and the results we obtain are more readily comparable to clinical observations. Using these substrates, we have found that MG63 osteoblast-like cells, normal human osteoblasts (NHOst cells), and fetal rat calvarial cells exhibit similar behaviors on tissue culture polystyrene (plastic) and smooth PT surfaces with respect to proliferation and differentiation [5–7]. In addition, they exhibit a decrease in proliferation and an increase in differentiation when treated with 1α,25(OH)2D3, that is typical of osteoblasts reported in the literature by many laboratories. 1α,25(OH)2D3 reduces cell number and stimulates osteocalcin production. When these cells are cultured on SLA and TPS, however, cell number is reduced and the effect of 1α,25(OH)2D3 on proliferation is less. Moreover, differentiation is markedly increased and it is further stimulated by 1α,25(OH)2D3.

This is also seen for factors related to osteoclast recruitment and activation. Messenger RNA transcripts for osteoprotegerin (OPG) and secreted OPG protein are both increased in MG63 cells cultured on SLA and TPS and are further increased by treatment of the cells with 1α,25(OH)2D3 [8]. In contrast, there is no change in RANK ligand (RANKL) mRNA or in the levels of soluble RANKL in the conditioned media of these cells.

MECHANISM OF 1α,25(OH)2D3 ACTION

These observations suggest that the change in phenotype caused by growth on the microrough substrates alters the response of osteoblasts to regulatory factors like 1α,25(OH)2D3. Osteoblasts grown on tissue culture plastic become more sensitive to 1α,25(OH)2D3 as they become more differentiated [9–11], but why this is the case is not well understood. The fact that osteoblasts exhibit a more differentiated phenotype when cultured on SLA and TPS provided us with an opportunity to investigate how sensitivity to 1α,25(OH)2D3 is regulated.

Our results indicate that part of the substrate effect is mediated by prostaglandin. MG63 cells produce low levels of PGE2 or PGE1 when cultured on plastic or smooth Ti substrates and this is unchanged when the cells are treated with 1α,25(OH)2D3 [12]. However, the amount of both prostanoids is increased when the cells are grown on SLA or TPS and treatment with 1α,25(OH)2D3 results in a synergistic increase in production [12]. Inhibition of prostanoid synthesis with indomethacin blocks the surface effect, including the response to 1α,25(OH)2D3. Specific inhibition of constitutive cyclooxygenase-1 (Cox-1) as well as specific inhibition of inducible Cox-2 also reduce the effects of surface roughness and 1α,25(OH)2D3, indicating that prostaglandin production via both pathways is required.

1α,25(OH)2D3 regulates osteoblasts through rapid protein kinase C (PKC) dependent signaling pathways mediated by the 1α,25(OH)2D3 membrane receptor ERp60 [11] as well as traditional nuclear vitamin D receptor (VDR) mechanisms [13], suggesting that one or both of these may have been affected. We are presently investigating whether either of these receptors has altered expression or if one or more components required for their action is/are changed by growth of the cells on microrough substrates.

ROLE OF INTEGRINS

One hypothesis is that growth on substrates with microrough structural feature alters integrin expression and function. Because integrins signal through some of the same pathways as 1α,25(OH)2D3, cross talk is possible. We found that mRNA levels for integrin subunits beta-1 (β1), alpha-2 and alpha-5, but not for alpha-v or beta-3, were affected by substrate roughness and that the effects were specific to each subunit and varied with time in culture [14]. 1α,25(OH)2D3 modulated levels of β1 and the effects were greater on the rougher surface. β1 is able to partner with alpha-2 and alpha-5, making it an excellent candidate for mediating the cross-talk between substrate-dependent integrin signaling and 1α,25(OH)2D3-dependent signaling.

We used several methods to investigate the role of the β1 integrin subunit. Our initial studies used antibodies to the β1 integrin to block signaling [15], as has been typically done in the literature [16; 17]. If antibodies are added to the culture medium together with the cells, they block adhesion. For this reason, MG63 cells were permitted to attach to the substrates and antibodies were added with the culture medium following attachment. Addition of anti β1 antibody AIIB2 to cells cultured on SLA and TPS had no significant effect on cell number and osteocalcin, but decreased alkaline phosphatase; MAB2253Z decreased cell number and alkaline phosphatase and increased osteocalcin in a dose-dependent manner. Effects of 1α,25(OH)2D3 on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased.

These results were initially confusing because they indicated that by blocking β1 integrin function, the cells were actually more differentiated and had an even greater response to 1α,25(OH)2D3. Related studies in our lab using PT and SLA substrates coated with polyethylene glycol and functionalized with an RGD-containing peptide demonstrated suggested that greater numbers of attachment sites might actually result in a less differentiated cell [18]. The behavior of MG63 cells on coated SLA could be restored to the behavior of these cells on PT and even tissue culture plastic simply by increasing the number of RGD peptides on the surface.

To overcome the limitations of the blocking antibody experimental design, we developed MG63 cell lines that stably expressed siRNA for the beta-1 integrin or a scrambled siRNA as a control [15]. β1-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor β1, prostaglandin E2, and osteoprotegerin in comparison with control cells. Moreover, β1-silencing inhibited the effects of surface roughness on these parameters.

While we anticipated that the response to surface microtopography would be affected, we were surprised to see that β1-silencing partially inhibited effects of 1α,25(OH)2D3. As shown in Figure 1a, β1 siRNA reduced the stimulatory effect of 1α,25(OH)2D3 on osteocalcin production on all surfaces. However, even though there was a marked reduction in osteocalcin production in cultures grown on SLA and TPS, the levels of this protein in the conditioned media was still elevated over that seen in cultures grown on plastic or PT. This was more evident when measuring OPG levels in the conditioned medium (Figure 1b). β1 siRNA reduced the stimulatory effect of 1α,25(OH)2D3 on all surfaces but the surface-dependent effect of 1α,25(OH)2D3 was only partially reduced on SLA and TPS.

Figure 1.

Figure 1

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Levels of osteocalcin (A) and OPG (B) in the conditioned media of confluent MG63 cells cultured on tissue culture polystyrene (plastic), smooth Ti, grit blasted/acid etched Ti (SLA), and Ti plasma sprayed surfaces (TPS). Cultures were treated with 10−8 M 1α,25(OH)2D3 (1,25) for 24 hours. Values are means + SEM for N = 6 independent cultures per variable. * p<0.05, Ti surface v. plastic surface; # p<0.05, with β1 siRNA v. without siRNA.

These results support the hypothesis that at least part of the interaction between surface topography and 1α,25(OH)2D3 on osteoblast differentiation is mediated by cross-talk between the two signaling pathways: one initiated by integrin function and one initiated by 1α,25(OH)2D3. A potential candidate for this is the ERK1/2 family of mitogen activated protein kinases. Integrin signaling can influence gene expression via ERK activation (5786) and we have shown that blocking activation of ERK1/2 with PD98059 affects osteoblast response to surface microstructure [19]. ERK1/2 also mediates some of the effects of 1α,25(OH)2D3 signaling via ERp60 [20] and others have shown that ERK1/2 participates in responses to 1α,25(OH)2D3 via the VDR [21]. Thus, by modifying osteoblast differentiation via changes in substrate topography, resulting changes in ERK1/2 may modify the actions of 1α,25(OH)2D3 through multiple mechanistic pathways.

Acknowledgments

The authors thank the funding agencies for this work including the National Science Foundation, the ITI Foundation, the Georgia Research Alliance, and the Price Gilbert, Jr. Foundation.

Footnotes

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