The authors have no conflict of interest.
Stretch-Induced PTH-Related Protein Gene Expression in Osteoblasts†
Article first published online: 28 MAR 2005
Copyright © 2005 ASBMR
Journal of Bone and Mineral Research
Volume 20, Issue 8, pages 1454–1461, August 2005
How to Cite
Chen, X., Macica, C. M., Ng, K. W. and Broadus, A. E. (2005), Stretch-Induced PTH-Related Protein Gene Expression in Osteoblasts. J Bone Miner Res, 20: 1454–1461. doi: 10.1359/jbmr.2005.20.8.1454
- Issue published online: 4 DEC 2009
- Article first published online: 28 MAR 2005
- Manuscript Accepted: 28 MAR 2005
- Manuscript Revised: 24 MAR 2005
- Manuscript Received: 2 AUG 2004
- PTH-related protein;
- mechanical loading;
- anabolic effect;
- ion channels
Mechanical forces play a critical role in regulating skeletal mass and structure. We report that mechanical loading induces PTHrP in osteoblast-like cells and that TREK-2 stretch-activated potassium channels seem to be involved in this induction. Our data suggest PTHrP as a candidate endogenous mediator of the anabolic effects of mechanical force on bone.
Introduction: Mechanical force has anabolic effects on bone. The PTH-related protein (PTHrP) gene is known to be mechanically inducible in smooth muscle cells throughout the organism, and N-terminal PTH and PTHrP products have been reported to have anabolic effects in bone. We explored the idea that PTHrP might be a candidate mediator of the effects of mechanical force on bone.
Materials and Methods: Mechanical loading was applied by swelling osteoblast-like cells in hypotonic solution and/or by application of cyclical stretch through a FlexerCell apparatus. RNase protection assay and real-time quantitative PCR analysis were used to assay PTHrP gene expression.
Results and Conclusion: Stretching UMR201-10B osteoblast-like cells by swelling in hypotonic solutions rapidly increased PTHrP mRNA. This induction was insensitive to gadolinium and nifedipine, to the removal of extracellular calcium, and to depletion of endoplasmic reticulum calcium, indicating that neither stretch-activated cation channels, L-type calcium channels, nor ER calcium is involved in the induction of PTHrP. The TREK family potassium channels are activated by both stretch and intracellular acidosis, and we identified these channels in osteoblast-like cells by PCR. Intracellular acidification increased PTHrP mRNA expression in UMR-201-10B cells, and siRNA targeted against the TREK-2 gene reduced endogenous TREK-2 expression and dampened PTHrP mRNA induction. Cyclical stretch also induced PTHrP in UMR-201-10B osteoblast-like cells and in MLO-A5 post-osteoblast-pre-osteocyte cells, the latter a stage in the osteoblastic differentiation program that is likely to be a key target of force in vivo. Our evidence suggests PTHrP as a candidate mediator of the anabolic effects of mechanical force on bone.
MECHANOTRANSDUCTION (MT) IS the term most often used to describe a cell's capacity to sense mechanical forces and translate them into a cellular response. Probably nowhere is this system of more fundamental importance than in the skeleton. Bone is sexually dimorphic, with the periosteal component being dominant in men and the endosteal component in women, and the principal factors that control periosteal and endosteal bone formation are mechanical force and estrogen, respectively.(1, 2) Given the fact that mechanical stimuli act directly on bone, it is increasingly important to define the local regulatory mechanisms of force in bone; these mechanisms are presently poorly understood.
PTH-related protein (PTHrP) was originally identified as the factor that is responsible for most instances of the syndrome of humoral hypercalcemia of malignancy (HHM).(3) The PTHrP gene is widely expressed in normal fetal and adult tissues, often in a hand-in-glove fashion with the type 1 PTH/PTHrP receptor, so-called because it appears to serve as a common receptor for the N-terminal domains of both PTH and PTHrP. Specificity of signaling thus seems to be based entirely on temporospatial considerations, PTH being a classical systemic peptide hormone and PTHrP a local regulatory product.(4)
We explored the idea that PTHrP might act as a local mediator of the effects of mechanical force on bone. This hypothesis was fashioned from four considerations: (1) PTHrP is known to be mechanically induced in smooth muscle cells in many sites (in which it acts in a short-loop feedback system to relax the smooth muscle structure in question(4); (2) N-terminal PTH and PTHrP products have similar if not identical anabolic effects in bone(5); (3) PTHrP is a normal product of cells of the osteoblastic lineage(6–9); and (4) conditional knock-out as well as haploinsufficiency of PTHrP in bone are associated with osteopenia,(10) seemingly prima facie evidence for a local regulatory effect of PTHrP in bone.
The cellular basis for the mechanical regulation of bone is envisioned as comprising both sensory and effector arms. The sensory component is made up of the neural-like network of osteocytes in cortical bone.(11) The effector component is made up of periosteal and endosteal lining cells in the outer and inner bone surfaces as well as contiguous endosteal cells of the osteoblastic lineage.(12–14) A number of intracellular mechanisms have been implicated in various stages of the loading response in mechanosensitive cells. These include stretch-activated cation (SA-cat) channels and voltage-gated calcium (L-VSCC) channels, both of which have been intensively studied in response to MT in bone cells in vitro.(15–18) Two-pore domain K+ channels of the TREK family (TREK-1, TREK-2, and TRAAK) are novel potassium channels that have been identified recently. TREK channels are strongly stimulated by mechanical pressure and are constitutively activated by intracellular acidosis. These channels are widely expressed in excitable cells but have not yet been studied in bone cells.(19)
In this report, we present evidence for a potential role for PTHrP in mediating the anabolic effects of mechanical force on bone.
MATERIALS AND METHODS
Cell culture and materials
The UMR-201–10B rat osteoblastic cell line was provided by Dr Kong Wah Ng (St Vincent's Hospital) and the MLO-A5 post-osteoblast/pre-osteocyte-like cell line was a gift from Dr Lynda Bonewald (University of Missouri, Kansas City, MO, USA). UMR-201–10B cells (passages 20–26) were grown in α-MEM medium supplemented with 10% FBS (Invitrogen) and 80 μg/ml gentamicin. MLO-A5 cell cultures were maintained in α-MEM medium supplemented with 5% FBS, 5% calf serum (CS; HyClone Laboratories, Logan, UT, USA), and antibiotics (penicillin/streptomycin; Invitrogen, Carlsbad, CA, USA). Incubation was carried out at 37°C in medium equilibrated with 5% CO2. All other chemicals were of analytical grade from Sigma unless otherwise noted.
Application of mechanical stimuli
Cells were subcultured until confluent on either rigid or flexible 6-well tissue culture plates coated with type 1 collagen. Hypotonic challenge was performed by replacing media with solutions varying in tonicity. The isotonic mannitol solution (317 mOsm) consisted of 154 mM mannitol, 65 mM NaCl, 5.5 mM KCl, 1 mM MgCl2, 1 mM CaC12, 20 mM HEPES (pH 7.4 with NaOH), 8 mM glucose, 1 mg/ml bovine serum albumin, and either 1 mM CaC12 or 0.2 mM EGTA (Ca2+-free solution). The hypotonic solutions (163, 240, 277, 300 mOsm) were similar except that the mannitol concentration was lower than that in the isotonic solution. Uniaxial cyclic stretch was performed with a FlexerCell strain unit (model FX-4000TM; Flexcell Corp., McKeesport, PA, USA). This system uses a vacuum to apply a defined deformation to cells attached to flexible-bottomed 6-well plates. Control plates were left unstrained for the same time period. Strain (0.8–1.0% elongation = 8000–10,000 μstrain) was applied with a frequency of 0.3 (3-s strain, 3-s rest) for 24 h. Results are expressed as percentages ± SD.
Intracellular pHi measurement
UMR-201–10B cells were cultured on glass coverslips for 2 days, transferred to a temperature-controlled perfusion chamber at 37°C, loaded with perfusate (isotonic mannitol) solution containing 5 μM BCECF-AM (Sigma) for 20 minutes, and kept in dye-free isotonic mannitol solution for 5 minutes before data were recorded. The intracellular pH (pHi) recording was performed on a perfusion chamber that was mounted on the stage of an inverted microscope (Diaphot; Nikon, Melville, NY, USA). BCECF was excited at 490 and 440 nm using a computer-controlled monochromator placed in front of a 75-W xenon lamp. Fluorescent images were obtained with an intensified charge-coupled video camera, with images obtained every 2 or 3 s. The 490/440 fluorescence intensity ratio was used to evaluate the relative pHi in different solutions. The emission wavelength was 535 nm.
Cell diameter measurement
The mean cell diameter of UMR-201–10B cells at various osmolarities was measured using a Coulter Counter (Beckman Coulter) per the manufacturer's instructions. Briefly, UMR-201–10B cells were cultured for 2 days and resuspended in mannitol solutions of various osmolarities for 1 h at 37°C before cell diameter measurement. The distribution curves were obtained, and mean cell diameter was calculated by computer software.
RNase protection assay
A subcloned 343-bp PvuII/BglII coding region fragment of the rat PTHrP cDNA was used to prepare the antisense RNA probe, which was hybridized at 2 × 105 cpm with 25 μg of total RNA. An antisense cyclophilin probe (1 × 104 cpm) was added to each sample to gauge internal loading.(20)
An siRNA sequence corresponding to nucleotide positions 692–710 of rat TREK-2 mRNA (accession AF385401) was selected according to the software provided by Ambion (Austin, TX, USA). An siRNA duplex with a fluorescein label at the 5′-end of the sense strand was synthesized by Dharmacon (Lafayette, CO, USA). Fluorescein-modified luciferase GL2 siRNA was used as negative control. Transfection of UMR-201–10B cells was carried out using Lipofectamine 2000 (Invitrogen). Immediately after transfection, siRNA+ cells were picked by FACS, recultured on 6-well plates coated with type 1 collagen, and allowed to recover in growth medium for 48 h before hypotonic challenge.
RT-PCR and quantitative real-time PCR analysis
Total RNA was extracted using an RNeasy Mini kit (Qiagen) followed by on-column RNase-free DNase I digestion per the manufacturer's instructions. Total RNA was reverse-transcribed using the StrataScript First-Strand RT-PCR kit (Stratagene). The cDNA product was used as template for PCR amplification using gene-specific primers that span intron 7 of rat TREK-2 genomic sequence (sense: 5′-GCAGCTGTCCTCAGTATGATTG-3′; antisense: 5′-TTAGTCCAGCTCCAGTGTC-3′). PCR conditions were initial denaturation at 94°C for 5 minutes; 35 cycles at 94°C for 45 s, 55°C for 1 minute, and 72°C for 2 minutes, and a final extension step at 72°C for 7 minutes. Real-time PCR was performed using DNA Engine Opticon 2 Continuous Fluorescence Detection System (MJ Research) with the following cycle parameters: 1 cycle of 95°C for 10 minutes followed by 40 cycles of 95°C for 15 s and 60°C for 1 minute. Analysis of the results was carried out using the Opticon 2 software supplied with the machine; the PTHrP readout was normalized against a β-actin internal control. The Taqman primer/probe sequences for rat PTHrP and β-actin are as follows (Applied Biosystems): PTHrP gene (M31603): sense, 5′-ACCATCTGATTGCGGAGATCCA-3′; antisense, 5′-TGTTGGGAGCAGGTTTGGAGTTAG-3′; probe, 6FAM-ATCAGAGCTACCTCGGAGGTGTCCC-MGBNFQ; rat β-actin gene (NM_03144), sense, 5′-AGAGGGAAATCGTGCGTGAC-3′; antisense, 5′-CGATAGTGATGACCTGACCGT-3′; probe, 6FAM-CACTGCCGCATCCTCTTCCTCCC-MGBNFQ.
All values are expressed as mean ± SD. ANOVA with subsequent Student's t-test was used to determine significant differences in multiple comparisons, p < 0.05 being considered significant.
Hypotonic induction of PTHrP mRNA in UMR-201–10B cells
UMR-201 cells(21) have abundant PTHrP and very little PTH/PTHrP receptor expression, making them a convenient system for studying PTHrP gene expression without potential feedback effects. We used osmotic induction of swelling/stretch as a surrogate for force by means of mannitol solutions of different tonicities, as described previously.(15) UMR-201–10B cells were treated with either isotonic or hypotonic solutions for different times (1.5–60 minutes), and total RNA was isolated from cells and assayed by RNase protection analysis. As shown in Fig. 1A, hypotonic treatment induced PTHrP mRNA in UMR-201–10B cells in a dose-dependent manner. From these data, we selected 240 mOsm as our routine hypotonic stimulus. The response was rapid, occurring as early as 30 minutes, peaking by 60 minutes (Fig. 1B), and plateauing over 4 h and beyond (data not shown). This rapid PTHrP mRNA response is reminiscent of PTHrP gene regulation in other sites and is typical of the kinetics of many cytokine-like local regulatory molecules.(22)
We used a Coulter Counter to measure the cell diameter in different solutions.(15) As shown in Fig. 1C, compared with the isotonic mannitol solution, hypotonic treatment at 240 mOsm for 1 h induced a 10% increase in diameter, whereas 277 mOsm yielded a 4.6% increase. Each experiment was repeated at least three times with similar results.
Calcium or calcium-related ion channels are not responsible for hypotonic induction of PTHrP
SA-cat and L-VSCC have been reported to be involved in the response of osteoblasts and osteocytes to osmotic and mechanical forces,(15–18) and calcium influx has been found to be the trigger for a number of downstream events on mechanical stimulation of osteogenic cells.(15, 16, 18) To test whether such channels are responsible for the hypotonic induction of PTHrP in UMR-201–10B cells, the SA-cat blocker, gadolinium, and the VSCC blocker, nifedipine, were added to the hypotonic solutions, either separately or in combination, and PTHrP expression was measured by RNase protection assay (RPA). As shown in Fig. 1D, the addition of gadolinium and/or nifedipine had no effect on PTHrP mRNA induction. Furthermore, removal of extracellular calcium or depletion of Ca2+ within the endoplasmic reticulum with thapsigargin also had no effect (Fig. 1E). These findings indicate that neither the SA-cat channel, the VSCC, nor extracellular or endoplasmic reticulum calcium seemed to be involved in the induction of PTHrP in response to hypotonicity.
TREK stretch-activated potassium channel expression in UMR-201–10B cells
The TREK family of two-pore domain potassium channels are novel stretch-activated channels that respond to stretch and/or intracellular acidosis.(19) To examine the expression of these channels in UMR-201–10B cells, we used RT-PCR followed by direct sequencing. Figure 2A shows RT-PCR products from UMR-201 cells as well as a comparison with the products from rat brain; these products had 100% sequence identity with the published rat TREK-2 sequence.
Using RPA, we found that intracellular acidification achieved by addition of sodium acetate to the medium increased PTHrP mRNA expression in a dose-dependent manner (Fig. 2B), consistent with regulation of PTHrP gene expression by the TREK-2 channel. To further define the role of TREK-2 stretch-activated potassium channels in osmotic regulation of PTHrP mRNA expression, RNA interference was performed by transfection of UMR-201 cells with fluorescein-labeled siRNA targeted to TREK-2, followed by cell sorting to isolate the siRNA+ cells. Real-time PCR was used to quantify mRNA readout. We found the expression of TREK-2 mRNA to be reduced by 80% 48 h after transfection (Fig. 2D), whereas the signal was unaffected in cells that either received nonsilencing control RNA or no siRNA (data not shown). In control cultures maintained in isotonic solution, PTHrP mRNA expression was not different between TREK-2 knock-down and control siRNA cultures, as expected. However, PTHrP mRNA expression in the hypotonic cultures was reduced by TREK-2 siRNA by 30% (Fig. 2E). These findings indicate that TREK-2 activity is involved in the hypotonic induction of PTHrP in UMR-201–10B cells but also suggest that other mechanosensitive channels such as volume-regulated anion channels(23) might be involved.
Use of uniaxial stretch as a mechanical stimulus and induction of PTHrP in cells at different stages of osteoblastic differentiation
To test whether PTHrP can be induced by direct mechanical stretch, we used the FlexerCell 4000 apparatus. We applied a 0.8–1.0% strain to UMR-201–10B cells for 24 h and found a 2-fold increase in PTHrP mRNA by real-time PCR (Fig. 3A). Because UMR-201 cells are regarded as examples of early osteoblastic or preosteoblastic cells,(21) whereas cells late in the osteoblastic differentiation program (e.g., late osteoblasts-lining cells-early osteocytes) are thought to be important physiological targets of strain in vivo,(24) we also examined MLO-A5 cells, which are phenotypically at the late osteoblast or pre-osteocyte stage.(25) These cells were tested with the same FlexerCell protocol and displayed a 3-fold increase in PTHrP mRNA by 24 h (Fig. 3B).
In this study, we provide initial evidence that PTHrP is a target of mechanical stimulation in osteoblastic cells. We show that mechanical loading induces PTHrP in osteoblast-like cells and that the TREK-2 stretch-activated potassium channel seems to be involved in this induction. Our data support PTHrP as a candidate endogenous mediator of the anabolic effects of mechanical force on bone.
There is some disagreement in the literature regarding the primacy of fluid sheer versus mechanical strain as the regulatory instrument of force in bone in vivo. How one approximates such stimuli in vitro is also a matter of debate. Osmotic approaches such as that used here have been widely used because of their simplicity and reproducibility, although they may stimulate channels other than those activated by mechanical forces per se and/or may also exert forces that are nonphysiological. Uniaxial systems such as FlexerCell units are also widely used, although these cannot reproduce the 3-D adhesive forces that characterize the actual microenvironment of bone cells in vivo and therefore must use higher than physiological levels of strain, as was the case here. In this study, we used both an osmotic approach and uniaxial stretching system and found that PTHrP is mechanically induced in both systems. The advantages and disadvantages of various approaches in vitro to the reproduction of force effects in vivo have been recently reviewed.(26)
It has been well documented that strain magnitude strongly influences bone cell responses on a tissue level, and it is generally accepted that 50–4000 μstrain is the range associated with physiological force regulation of bone cell activities in vivo.(27) In our study, the amount of strain applied to the cells (8000–10,000 μstrain) to stimulate PTHrP gene upregulation is higher than these values, and we have not been able to detect PTHrP gene activation under 1000–5000 μstrain. This finding agrees with the notion that mechanical strain at physiological levels in bone tissue may not stimulate a response in bone cells in vitro,(28) for which reason many groups use fluid shear as their studies in vitro.(26) The swelling we induced in the hypotonic experiments was likely associated with nonphysiological levels of strain as well, although the degree of swelling of the immobilized cells in culture was probably considerably less than the degree of swelling measured in cells in suspension.(29)
Stretch duration or cycle number is another important aspect of bone MT, and in our study, we found a moderate but stable induction of PTHrP on 4-h (2400 cycles) mechanical stretch, which became obvious after 24 h (14,400 cycles). The PTHrP gene has the regulatory features of a primary response gene and may display either early and transient or a persistent response in different cell types and/or in response to different stimuli.(30, 31) The PTHrP mRNA response was rapid in the hypotonic experiment and relatively delayed in the Flexercell experiments. At present, we do not have an explanation for the different kinetic responses of PTHrP mRNA in these two systems. However, if the PTHrP gene is deployed as a component of an anabolic bone cell response program after force induction, the gene might well not part of the immediate response in the force-induced cascade. The experiments described in this study were all preformed at 0.3 Hz and therefore did not address the potential frequency-dependent effects of stretch on PTHrP gene activation. This is an important variable that we have not yet examined.
Our experiments were performed almost exclusively with two established cell lines: one thought to represent an early(21) and the other a later phenotype(24) in the osteoblast differentiation program. In preliminary Flexercell experiments, we found a moderate (∼50%) PTHrP mRNA response in primary calvarial cells cultured from newborn mice. Because of this modest response and the inconvenience of the system, we used the cell lines in the remainder of our experiments.
In the hypotonic model, we identified a possible role for the TREK-2 channel in mediating hypotonic induction of PTHrP gene expression. Given that we could show that this channel only accounts for some 30% of the induction, other mechanisms may be involved. In preliminary studies, we detected several candidate genes for volume-activated chloride channels in UMR-201–10B cells, and we also found that hypotonic induction of PTHrP was impaired when chloride channel blockers such as DIDS and SITS were added to the hypotonic mannitol solution. This work is ongoing. In a preliminary study, we also detected the expression of TREK genes in MLO-A5 cells and primary cultured murine calvarial cells (data not shown).
The two-pore TREK family is the most recently described family of K+ channels. These channels are widely distributed in the central and peripheral nervous systems, and some family members are expressed in nonexcitable cells as well. TREK channels are voltage insensitive and contribute to the background K+ currents that set the resting membrane potential, so that modulation of their activity can influence the excitability of the cell in question. They are insensitive to classical K+ channel blockers and are regulated by a number of factors or environmental changes such as mechanical stretch/cell volume, pH, and fatty acid concentrations. These channels have not been previously identified and/or studied in cells of the osteoblastic lineage, but their properties (particularly regarding resting membrane potential) would appear to make them well suited to participate in the regulation of bone by mechanical forces.
One well-studied function of PTHrP is as a developmental regulatory molecule in a variety of different systems.(4) An evolving theme of PTHrP function in the adult is the increasing recognition that PTHrP is responsible for regulatory effects that in earlier days were attributed to PTH itself. A case in point is PTHrP regulation of excitable cells such as neurons and smooth muscle.(4, 32) For example, PTHrP seems to be expressed in every smooth muscle cell in the organism, in which the gene seems to be stretch-induced, providing a PTHrP-dependent short-loop relaxation system that allows the smooth muscle bed or structure in question to accommodate pressure or gradual filling.(4) This pattern of stretch induction supplied a lead for the work described here, in which we report force induction of PTHrP in osteoblastic bone cells and propose that PTHrP may be a mediator of the anabolic effects of mechanical forces in the skeleton. Whereas PTH itself has well-known anabolic effects in the skeleton(33) (and is in fact marketed as an anabolic agent for the treatment of osteoporosis), it constitutes a classical systemic peptide hormone that is secreted as a direct function of the ambient calcium concentration and thus is not a viable candidate as a mediator of the local regulatory effects of MT in bone. We suggest that PTHrP is a candidate as such a mediator. Bone may actually represent a site in which evolution has created domains of influence for both PTH and PTHrP, possibly even with the potential for overlap under certain biological conditions.
The authors thank Drs William Philbrick and John Wysolmerski for critical review of the work, Dr Mark Horowitz for use of the Flexercell apparatus, Dr Jonathan Bogan for guidance on siRNA, Dr Lynda Bonewald for providing the MLO-A5 cell line, and Barbara Dreyer, Guoying Liang, and Oindrilla Chatterjee for expert technical assistance. Dr John Geibel and Stephanie Busqueand provided guidance and assistance in the intracellular pH measurement. Yale Core Center for Musculoskeletal Disorders Grant AR 46032 provided access to the Opticon-2 real-time PCR machine. This work was supported by NIH Grants DK 62515 and DK 48108.
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