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GROWTH, DIFFERENTIATION, AND APOPTOSIS

Human adipose-derived adult stem cells upregulate palladin during osteogenesis and in response to cyclic tensile strain

¹Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh; and ²Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

Submitted 16 February 2007 ; accepted in final form 6 August 2007

	ABSTRACT

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Cell morphology may be an important stimulus during differentiation of human adipose-derived adult stem (hADAS) cells, but there are limited studies that have investigated the role of the cytoskeleton or associated proteins in hADAS cells undergoing differentiation. Palladin is an actin-associated protein that plays an integral role in focal adhesion and cytoskeleton organization. In this study we show that palladin was expressed by hADAS cells and was modulated during osteogenic differentiation and in response to cyclic tensile strain. Human ADAS cells expressed the 90- and 140-kDa palladin isoforms and upregulated expression of both isoforms after culture in conditions that promoted osteogenesis. Palladin mRNA expression levels were also increased in hADAS cells subjected to cyclic tensile strain. Knockdown of the palladin gene during osteogenesis resulted in decreased actin stress fibers and decreased protein levels of Eps8, an epidermal growth factor receptor tyrosine kinase that colocalizes with actin. Silencing the palladin gene, however, did not affect hADAS cells' commitment down the osteogenic lineage.

adipose-derived adult stem cells; mechanobiology; Eps8; actin cytoskeleton; mesenchymal stem cells

Palladin is a member of the palladin-myotilin-myopalladin gene family (19). There are three palladin isoforms that are most commonly found in both mouse and human tissues. The 90- to 92-kDa isoform is the most abundant isoform in cells and is ubiquitously expressed in most embryonic tissues. The 140-kDa isoform is also expressed in most embryonic tissues and in adult smooth muscle tissues. The 200-kDa isoform expression is limited primarily to the heart and bone (17, 20, 22). Palladin binds to multiple actin-associated proteins, including vasodilator-stimulated phosphoprotein (3), -actinin (20, 25), ezrin (17), Lasp-1 (22), profilin (2), ArgBP2 (24), and Eps8 (9). In addition to regulating normal actin cytoskeleton formation, palladin also regulates focal adhesion formation, and thus modulates cell morphology and migration (20).

The purpose of this study was to investigate the presence of palladin in hADAS cells and the effects of osteogenic differentiation and mechanical load on palladin expression levels. It was hypothesized that hADAS cells would express palladin and that osteogenic differentiation and cyclic tensile strain would upregulate palladin expression levels. It was also hypothesized that knockdown of palladin would alter the actin cytoskeleton organization and thus alter osteogenic differentiation.

	MATERIALS AND METHODS

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Cell isolation, culture, and characterization. Excess human adipose tissue from abdominoplasty procedures was obtained from three female donors (45-year-old African American, 31-year-old Caucasian, and 35-year-old Caucasian) in accordance with an approved IRB protocol. Human ADAS cells were isolated from the tissue using a method based on density and differential adhesion, as described by Zuk et al. (30). Briefly, adipose tissue was digested with 0.075% collagenase type I (Worthington Biochemical, Lakewood, NJ) for 30 min, and then the ADAS cell-rich dense cell fraction was pelleted by centrifugation at 12,000 g. Blood cells were lysed by incubation and resuspension of the pelleted cells in 160 mM NH₄Cl. Stromal cells were then pelleted by centrifugation at 12,000 g for 10 min and then resuspended in -MEM supplemented with 10% fetal bovine serum (FBS) (lot selected; Atlanta Biologicals, Lawrenceville, GA), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (growth medium). After 24 h, the cell monolayer was washed twice with phosphate-buffered saline to remove nonadherent cells, and fresh growth medium was added. Human ADAS cells were then characterized via immunohistochemical analysis of surface markers that have been found to be present (CD73, CD105, and CD166) and absent (CD34 and CD45) in hADAS cells and by their ability to differentiate down osteogenic and adipogenic pathways. All cell culture chemicals and supplies were purchased from Mediatech (Herndon, VA) and GIBCO-BRL (Grand Island, NY) unless otherwise noted.

Osteogenic differentiation. Cells were plated at 50,000–100,000 cells/10 cm² and grown until they reached 100% confluency. Cells were then cultured for 2 wk in growth or osteogenic medium. Osteogenic medium consisted of growth medium supplemented with 50 µM ascorbic acid, 0.1 µM dexamethasone, and 10 mM β-glycerolphosphate (5, 10–11, 18, 27). Extent of osteogenic differentiation was determined by deposition of calcium. Calcium deposits were visualized by staining with Alizarin Red S.

Fabrication of collagen gels. Human ADAS cells were seeded into collagen gels consisting of 70% type I collagen (BD Biosciences, San Jose, CA) (pH adjusted to 7.0), 20% 5x MEM, and 10% FBS at 60,000 cells/200 µl gel solution. The cell-seeded gel solutions were loaded into TissueTrain collagen I-coated six-well culture plates (Flexcell International, Hillsborough, NC) to create linear three-dimensional collagen constructs.

Application of tensile strain. Cell-seeded constructs were subjected to 14 days of 10% cyclic uniaxial tensile strain at 1 Hz for 4 h/day using a Tissue Train Flexcell Strain Unit (FX-4000, Flexcell International).

RNA isolation and real-time RT-PCR analysis. Total RNA was purified using Eppendorf Perfect RNA mini-columns (Hamburg, Germany) according to the manufacturer's recommended protocol for eukaryotic cells. Total RNA was quantitated using a microplate-based RiboGreen method (Molecular Probes, Eugene, OR). A single pool of cDNA was reverse transcribed from about 15–300 ng of each RNA sample using SuperScript III (Invitrogen, Carlsbad, CA) with oligo dT primers. Real-time PCR was performed using an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). TaqMan-based PCR Assays-on-Demand (Applied Biosystems) were used for gene expression analysis of palladin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the endogenous control. Expression levels were determined with the C_T method (14).

Immunohistochemical analysis. Cells were fixed with 10% formalin. Cell membranes were permeabilized using 0.2% Triton X-100 and 0.5% BSA. Three primary antibodies were utilized with specificity to different palladin isoforms. These antibodies included a mouse monoclonal anti-palladin antibody against all isoforms (IE6) (20), a rabbit polyclonal anti-palladin antibody against all isoforms, and a rabbit polyclonal anti-palladin antibody against the 140- and 200-kDa isoforms (4IgNT) (22). Cells were counterlabeled with appropriate secondary polyclonal antibodies conjugated with either Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes) to visualize palladin. Cells were also stained with Alexa Fluor 594 phalloidin (Molecular Probes) and DAPI to label f-actin and nuclei, respectively. Cells were viewed at room temperature with a Leica DM LFSA epifluorescent microscope (Wetzlar, Germany) equipped with a x40 water immersion, UV objective lens; a Hamamatsu ORCA ERG deep-cooled, high-resolution camera (Hamamatsu City, Japan); and SimplePCI image capture and analysis software (Compix, Sewickley, PA).

Western blot analysis. Cells were collected by scraping into a lysis buffer (0.5% DOC, 0.1% SDS, 0.1% Nonidet-40, 10 mM EDTA, 150 mM KCl, 20 mM HEPES, pH 7.4, with the Sigma protease inhibitor cocktail for mammalian cells). Lysates were gently rotated at 4°C and then centrifuged for 15 min at 15,000 g. Supernatants were collected and protein concentrations calculated by BCA assay (Pierce, Rockford, IL). Supernatants were then boiled for 5 min in Laemmli sample buffer, resolved on 4–12% 1.0 mm Tris-Bis acrylamide gels (Invitrogen), and electroblotted onto nitrocellulose. The membrane was stained with Ponceau S to check for correct protein loading; blocked overnight in 2% gelatin in Tris-buffered saline (TBS); and stained with monoclonal anti-tubulin (1:10,000; Chemicon, Temecula, CA), monoclonal anti-actin (1:10,000; Sigma, St. Louis, MO), and rabbit anti-palladin serum (1:5,000) in TBS with 0.05% Tween. After 1 h of incubation, the membrane was washed three times with TBS-Tween and then incubated for an hour with IRdye700 and IRdye800 infrared-tagged anti-mouse (1:20,000) and anti-rabbit (1:15,000) secondary antibodies. The membrane was then washed three times in TBS-Tween and then briefly washed in TBS before being imaged with the infrared imaging Odyssey scanner (LI-COR Biosciences; Lincoln, NE). Protein levels were determined using LI-COR's Odyssey Application software version 1.2, after background levels were corrected for using the "median; right and left" setting. This analysis provided arbitrary intensity units for each band (tubulin, actin, and palladin).

siRNA knockdown of palladin. Small interfering RNA (siRNA) duplex 19-base oligonucleotides were purchased from Dharmacon Research (Lafayette, CO) (target sequence: UCACUACACCAUUCAAAGA). The control was siCONTROL Non-Targeting siRNA #1 (Dharmacon). On day 0, cells were transfected using the transfection reagent TransIT siQuest (Mirus Bio, Madison, WI). Two wells from each donor received 50 nM of either the palladin or control siRNA. On day 2, six wells from each treatment group were switched to osteogenic medium. On day 9, the siRNA transfection was repeated by using the same conditions as on day 0. On day 16, the cells were harvested, and protein was collected for Western blot analysis or RNA was harvested for RT-PCR analysis.

Statistical analysis. For mRNA and protein expression levels, data were subjected to a two-tailed Student's t-test or rank sum t-test to determine significant differences between control and osteogenic conditions on appropriate days (P < 0.05). To determine significant differences among hADAS cells subjected to both mechanical and chemical stimuli, data were subjected to a one-way ANOVA followed by a two-tailed Student's t-test (P < 0.05). Data are presented as means ± SD.

	RESULTS

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Western blot results indicated that hADAS cells in monolayer culture, which were positive for the stem cell markers CD105 and CD166, constitutively expressed the 90- and 140-kDa palladin isoforms but not the 200-kDa isoform (Fig. 1, A and B). Immunohistochemical staining with phalloidin and either an antibody against all three palladin isoforms or an antibody against the two larger isoforms showed that palladin was primarily present in the cells in linear bundles (Fig. 1, C and D) and occasionally as punctate localizations within the cytoplasm (Fig. 2B). Palladin bundles were located throughout the cytoplasm and along actin-rich stress fibers (Fig. 1, C and D). To determine whether the 90- and 140-kDa isoforms were located within similar regions of the cells, hADAS cells were double labeled with an antibody against both palladin isoforms or an antibody against the 140-kDa isoform. Human ADAS cells had colocalized expression of both the 90- and 140-kDa palladin isoforms (Fig. 2A). However, the 90-kDa palladin isoform was not always colocalized with the 140-kDa isoform (Fig. 2B), and some cells expressed primarily the 90-kDa but not the 140-kDa isoform (Fig. 2C).

View larger version (82K): [in this window] [in a new window]   Fig. 1. Human adipose-derived adult stem (ADAS) cells express palladin. A and B: Western blot analyses of human ADAS cells from 3 donors labeled with a monoclonal antibody against all palladin isoforms (A) or a polyclonal antibody against the 140- and 200-kDa isoforms (B). C and D: images of human ADAS cells labeled with the monoclonal (C) or the polyclonal palladin (D) antibody (green). Red is phalloidin-stained actin filaments, and blue is DAPI-stained nuclei. Scale bar is 100 µm.

View larger version (99K): [in this window] [in a new window]   Fig. 2. Localization of various palladin isoforms. Human ADAS cells labeled with the IE6 antibody (red) to visualize all palladin isoforms, the 4IgNT antibody (green) to visualize only the 140-kDa isoform, and DAPI (blue) to visualize the nuclei. A: hADAS cells have colocalization of both the 90- and 140-kDa isoforms. B: 90-kDa isoform does not always colocalize with the 140-kDa isoform. C: hADAS cells primarily expressing the 90-kDa isoform. Scale bar is 100 µm.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Increased palladin expression during osteogenesis and mechanical load. A: fold increase in palladin mRNA expression levels in hADAS cells in monolayer after culture in growth or osteogenic media for 1, 7, or 14 days. All mRNA expression levels were normalized to GADPH levels. n = 3 donors with 3 replicates/donor. B: fold increase in palladin mRNA expression levels in hADAS cells seeded in three-dimensional type I collagen gels and cultured in growth or osteogenic media for 1, 7, or 14 days and subjected to 10% uniaxial tensile strain at 1 Hz for 4 h/day. n = 2 donors with 1–3 replicates/donor. C: Western blot image of hADAS cells in monolayer after culture in growth (G) or osteogenic (O) media for 1, 7, or 14 days. D: relative change in protein expression of 90-kDa palladin after 1, 7, or 14 days of culture in growth or osteogenic media. Protein levels were normalized to actin levels. Values are means ± SD. **P < 0.001, *P < 0.05, #P < 0.1.

To determine whether palladin could affect actin organization in hADAS cells, the palladin gene was silenced by siRNA treatment. Cells were then cultured for 3 days in conditions that promoted osteogenesis, and the cytoskeleton was labeled with phalloidin and viewed by indirect immunofluorescence. There was no observable difference seen in the actin cytoskeleton organization between hADAS cells in growth media and those cells in osteogenic media (Fig. 4). Cells appeared to have long actin stress fibers that extended the length of the cell body. Knockdown of palladin appeared to decrease the number of long stress fibers within hADAS cells in both growth and osteogenic media as determined by visual assessment (Fig. 4). These cells had shorter actin bundles near the cell periphery (Fig. 4, arrow) and less definable stress fibers throughout the cell body (Fig. 4, arrowhead). However, these differences in cytoskeletal organization did not alter the ability of the cells to undergo osteogenesis since silencing the palladin gene did not alter calcium deposition or protein levels of osteopontin after 2 wk of culture in osteogenic media (Fig. 5).

View larger version (58K): [in this window] [in a new window]   Fig. 4. Knockdown of palladin alters f-actin expression. A and B: images of untreated hADAS cells and hADAS cells transfected with a control small interfering RNA (siRNA) or palladin siRNA and cultured in growth (A) or osteogenic media (B) for 3 days. hADAS cells transfected with palladin siRNA had shorter actin bundles near the cell periphery (arrows) and less definable stress fibers throughout the cell body (arrowheads). Red is phalloidin-stained actin filaments, and blue is DAPI-stained nuclei. Scalebar is 100 µm.

View larger version (68K): [in this window] [in a new window]   Fig. 5. Knockdown of palladin does not affect osteogenesis. A: Alizarin red S-stained calcium deposits in cultures of hADAS cells transfected with a control siRNA or palladin siRNA and cultured in osteogenic media for 14 days. Cells counterstained with hematoxylin. Scale bar is 100 µm. B: relative change in osteopontin protein expression in hADAS cells transfected with a control siRNA or palladin siRNA and cultured in osteogenic media for 14 days. Values were normalized to tubulin.

View larger version (58K): [in this window] [in a new window]   Fig. 6. Knockdown of palladin alters actin associated protein expression. A: Western blot image of 90-kDa palladin, Eps8, and tubulin expression in normal hADAS cells (lanes 1 and 2) and hADAS cells transfected with a control siRNA (lane 3) or palladin siRNA (lane 4). Cells were cultured in growth media (lane 1) or osteogenic media (lanes 2–4) for 14 days. B: relative change in protein expression of 90-kDa palladin and Eps8. Values were normalized to tubulin. *P < 0.05.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

Palladin was found to be upregulated in the hADAS cells during both osteogenesis and tensile strain. Furthermore, silencing the palladin gene decreased actin stress fibers, but it did not affect the ability of the cells to undergo osteogenesis. hADAS cells were still able to deposit calcium, and protein levels of osteopontin were not affected by palladin knockdown. Contrastingly, Rodriguez et al. (23) found that disruption of the actin cytoskeleton with cytochalasin D decreased calcium deposition and alkaline phosphatase activity in bone marrow-derived mesenchymal stem cells undergoing osteogenesis. Since the knockdown of palladin did not completely disrupt the cytoskeleton, and prior studies indicate that tension in the cytoskeleton affects stem cell lineage commitment (15), our data suggest that knockdown of palladin did not alter cytoskeletal tension enough to affect the ability of hADAS cells to undergo osteogenesis in response to chemical factors.

Knockdown of palladin did decrease Eps8 protein levels. Eps8 is an unusual protein that affects the actin cytoskeleton through two distinct pathways. First, Eps8 was shown to be part of a trimeric complex that activates the small GTPase Rac and thus stimulates the formation of actin-rich membrane ruffles (13, 26). In addition to this indirect pathway for influencing actin organization, Eps8 has also been shown to bind directly to actin filaments and cap their rapidly growing ends (6, 7). Thus Eps8 can play a direct role in organizing actin filament arrays in which the filaments are all the same length. The fact that palladin knockdown in stem cells resulted in decreased levels of Eps8 suggests that binding of palladin may increase the half-life of Eps8 in cells, a possibility that would have to be explored in future studies.

This study found that palladin was upregulated in hADAS cells subjected to cyclic tensile strain. Furthermore, there was an additive effect on palladin mRNA expression levels in hADAS cells cultured in osteogenic media while being subjected to tensile strain. Mechanical load can alter actin cytoskeleton organization (16). Additionally, cytoskeletal interactions can modulate the response of bone cells to mechanical signals (21, 29). To this end, cytoskeletal interactions could thus affect the response of hADAS cells to mechanical load. Recently, Engler et al. (8) demonstrated that naive mesenchymal stem cells sense the mechanical properties of the environment, and that this is a major determinant of cell fate. The authors showed that the level of organization of the actin cytoskeleton and the degree of cell contractility (which is determined by the degree of stiffness of the extracellular matrix) drove stem cells to follow a neurogenic, myogenic, or osteogenic lineage. Mechanical signals can be detected by the cells through interactions among the matrix, integrins, and the cytoskeleton (1). If palladin affected these interactions, then palladin could modulate the response of the cell to mechanical load. However, this study only indicates that cyclic strain can upregulate palladin mRNA levels and does not indicate whether palladin can affect mechanotransduction in hADAS cells. Further studies would need to be conducted to determine this latter point.

This study is the first to show that palladin is present in hADAS cells and that palladin was upregulated in hADAS cells during osteogenic differentiation and during mechanical load. Although the function of palladin within hADAS cells is unknown, knockdown of palladin was found to decrease actin stress fibers and Eps8 expression without affecting the ability of hADAS cells to differentiate down the osteogenic pathway.

	GRANTS

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This study was supported by the Ralph E. Powe Faculty Enhancement Award (to E. G. Loboa), National Institutes of Health Grants NS-43243 and GM-61743 (to C. A. Otey), and a North Carolina Biotechnology Center Multidisciplinary Research Grant (to E. G. Loboa).

	ACKNOWLEDGMENTS

 
The authors thank Drs. W. Losken and J. van Aalst for providing the adipose tissue samples and Dr. S. Bernacki, D. Arneman, A. Finger, S. Goicoechea, A. Hanson, C. Haslauer, and P. Middlebrooks for technical assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: E. G. Loboa, Joint Dept. of Biomedical Engineering at UNC-Chapel Hill and NC State Univ., 2142 Burlington Laboratories, Campus Box 7115, NC State Univ., Raleigh, NC 27695-7115 (e-mail: egloboa{at}ncsu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^* M. E. Wall and A. Rachlin contributed equally to this study.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
1. Banes AJ, Lee G, Graff R, Otey C, Archambault J, Tsuzaki M, Elfervig M, Qi J. Mechanical forces and signaling in connective tissue cells: cellular mechanisms of detection, transduction, and responses to mechanical deformation. Current Opin Orthopedics 12: 389–396, 2001.[CrossRef]

2. Boukhelifa M, Moza M, Johansson T, Rachlin A, Parast M, Huttelmaier S, Roy P, Jockusch BM, Carpen O, Karlsson R, Otey CA. The proline-rich protein palladin is a binding partner for profilin. FEBS J 273: 26–33, 2006.[CrossRef][Medline]

3. Boukhelifa M, Parast MM, Bear JE, Gertler FB, Otey CA. Palladin is a novel binding partner for Ena/VASP family members. Cell Motil Cytoskeleton 58: 17–29, 2004.[CrossRef][Web of Science][Medline]

4. Boukhelifa M, Parast MM, Valtschanoff JG, LaMantia AS, Meeker RB, Otey CA. A role for the cytoskeleton-associated protein palladin in neurite outgrowth. Mol Biol Cell 12: 2721–2729, 2001.[Abstract/Free Full Text]

5. Colter DC, Sekiya I, Prockop DJ. Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci USA 98: 7841–7845, 2001.[Abstract/Free Full Text]

6. Croce A, Cassata G, Disanza A, Gagliani MC, Tacchetti C, Malabarba MG, Carlier MF, Scita G, Baumeister R, Di Fiore PP. A novel actin barbed-end-capping activity in EPS-8 regulates apical morphogenesis in intestinal cells of Caenorhabditis elegans. Nat Cell Biol 6: 1173–1179, 2004.[CrossRef][Web of Science][Medline]

7. Disanza A, Carlier MF, Stradal TE, Didry D, Frittoli E, Confalonieri S, Croce A, Wehland J, Di Fiore PP, Scita G. Eps8 controls actin-based motility by capping the barbed ends of actin filaments. Nat Cell Biol 6: 1180–1188, 2004.[CrossRef][Web of Science][Medline]

8. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell 126: 677–689, 2006.[CrossRef][Web of Science][Medline]

9. Goicoechea S, Arneman D, Disanza A, Garcia-Mata R, Scita G, Otey CA. Palladin binds to Eps8 and enhances the formation of dorsal ruffles and podosomes in vascular smooth muscle cells. J Cell Sci 119: 3316–3324, 2006.[Abstract/Free Full Text]

10. Halleux C, Sottile V, Gasser JA, Seuwen K. Multi-lineage potential of human mesenchymal stem cells following clonal expansion. J Musculoskelet Neuronal Interact 2: 71–76, 2001.[Medline]

11. Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 64: 295–312, 1997.[CrossRef][Web of Science][Medline]

12. Kaibuchi K, Kuroda S, Amano M. Regulation of the cytoskeleton and cell adhesion by the Rho family GTPases in mammalian cells. Annu Rev Biochem 68: 459–486, 1999.[CrossRef][Web of Science][Medline]

13. Lanzetti L, Rybin V, Malabarba MG, Christoforidis S, Scita G, Zerial M, Di Fiore PP. The Eps8 protein coordinates EGF receptor signalling through Rac and trafficking through Rab5. Nature 408: 374–377, 2000.[CrossRef][Medline]

14. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(–C(T)) method. Methods 25: 402–408, 2001.[CrossRef][Web of Science][Medline]

15. McBeath R, Pirone DM, Nelson CM, Bhadriraju K, Chen CS. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell 6: 483–495, 2004.[CrossRef][Web of Science][Medline]

16. Meazzini MC, Toma CD, Schaffer JL, Gray ML, Gerstenfeld LC. Osteoblast cytoskeletal modulation in response to mechanical strain in vitro. J Orthop Res 16: 170–180, 1998.[CrossRef][Web of Science][Medline]

17. Mykkanen OM, Gronholm M, Ronty M, Lalowski M, Salmikangas P, Suila H, Carpen O. Characterization of human palladin, a microfilament-associated protein. Mol Biol Cell 12: 3060–3073, 2001.[Abstract/Free Full Text]

18. Noth U, Osyczka AM, Tuli R, Hickok NJ, Danielson KG, Tuan RS. Multilineage mesenchymal differentiation potential of human trabecular bone-derived cells. J Orthop Res 20: 1060–1069, 2002.[CrossRef][Web of Science][Medline]

19. Otey CA, Rachlin A, Moza M, Arneman D, Carpen O. The palladin/myotilin/myopalladin family of actin-associated scaffolds. Int Rev Cytol 246: 31–58, 2005.[Web of Science][Medline]

20. Parast MM, Otey CA. Characterization of palladin, a novel protein localized to stress fibers and cell adhesions. J Cell Biol 150: 643–656, 2000.[Abstract/Free Full Text]

21. Pavalko FM, Chen NX, Turner CH, Burr DB, Atkinson S, Hsieh YF, Qiu J, Duncan RL. Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions. Am J Physiol Cell Physiol 275: C1591–C1601, 1998.[Abstract/Free Full Text]

22. Rachlin AS, Otey CA. Identification of palladin isoforms and characterization of an isoform-specific interaction between Lasp-1 and palladin. J Cell Sci 119: 995–1004, 2006.[Abstract/Free Full Text]

23. Rodriguez JP, Gonzalez M, Rios S, Cambiazo V. Cytoskeletal organization of human mesenchymal stem cells (MSC) changes during their osteogenic differentiation. J Cell Biochem 93: 721–731, 2004.[CrossRef][Web of Science][Medline]

24. Ronty M, Taivainen A, Moza M, Kruh GD, Ehler E, Carpen O. Involvement of palladin and alpha-actinin in targeting of the Abl/Arg kinase adaptor ArgBP2 to the actin cytoskeleton. Exp Cell Res 310: 88–98, 2005.[CrossRef][Web of Science][Medline]

25. Ronty M, Taivainen A, Moza M, Otey CA, Carpen O. Molecular analysis of the interaction between palladin and alpha-actinin. FEBS Lett 566: 30–34, 2004.[CrossRef][Web of Science][Medline]

26. Scita G, Tenca P, Areces LB, Tocchetti A, Frittoli E, Giardina G, Ponzanelli I, Sini P, Innocenti M, Di Fiore PP. An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine. J Cell Biol 154: 1031–1044, 2001.[Abstract/Free Full Text]

27. Sottile V, Halleux C, Bassilana F, Keller H, Seuwen K. Stem cell characteristics of human trabecular bone-derived cells. Bone 30: 699–704, 2002.[Medline]

28. Sumanasinghe RD, Bernacki SH, Loboa EG. Osteogenic differentiation of human mesenchymal stem cells in collagen matrices: effect of uniaxial cyclic tensile strain on bone morphogenetic (BMP-2) mRNA expression. Tissue Eng 12: 3459–3465, 2006.[CrossRef][Web of Science][Medline]

29. Toma CD, Ashkar S, Gray ML, Schaffer JL, Gerstenfeld LC. Signal transduction of mechanical stimuli is dependent on microfilament integrity: identification of osteopontin as a mechanically induced gene in osteoblasts. J Bone Miner Res 12: 1626–1636, 1997.[CrossRef][Web of Science][Medline]

30. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7: 211–228, 2001.[CrossRef][Web of Science][Medline]

This Article Abstract Full Text (PDF) All Versions of this Article: 293/5/C1532    most recent 00065.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wall, M. E. Articles by Loboa, E. G. Search for Related Content PubMed PubMed Citation Articles by Wall, M. E. Articles by Loboa, E. G.