The expression of two collagen receptor subfamilies,integrins and discoidin domains during osteogenic and chondrogenic differentiation of human mesenehymal stem cells

Abstract

BACKGROUND:

Collagen receptors are characterized by binding to and being activated by collagens. We know little about the molecular mechanism by which the integrins and discoidin domains (DDRs) recognize collagen.

OBJECTIVE:

The aim of this study was to investigate the expression of two main collagen receptor subfamilies, integrins and DDRs, during osteogenic and chondrogenic differentiation of human mesenehymal stem cells (hMSCs).

METHODS:

Using qRT-PCR, Western blots and FACS, the levels of DDR1, DDR2, integrin subunits β1, α1, α2, α10 and α11 receptors on hMSCs, were assessed upon activation by collagen type I, as well as during osteogenic and chondrogenic differentiation.

RESULTS:

The expression of DDR2 and integrin α11β1 was altered compared with other receptors when the cells were cultured under undifferentiated conditions. During osteogenic and chondrogenetic differentiation, DDR2 and α11 were up-regulated during early stages (6 day) of osteogenesis and chondrogenesis, respectively. The expression and activation of DDR2 was concomitant with another receptor integrin subunit β1 during osteogenetic differentiation.

CONCLUSIONS:

The results suggested that DDR2 was more specific for osteogenesis than chondrogenesis, while integrin α11β1 was more specific in chondrogenesis. DDR2 and α11 may play a role in the regulation of osteogenesis and chondrogenesis based on the differential expression of these receptors during lineage-dependent changes.

Keywords

Collagen receptors human mesenehymal stem cell osteogenesis chondrogenesis

1. Introduction

The most widely expressed mammalian receptors for collagen are four members of the β1 integrin family and the discoidin domain receptors (DDRs). Integrins are a major class of the extracellular matrix (ECM) receptors for cell adhesion [1]. They are heterodimeric transmembrane glycoproteins that are composed of non-covalently associated α and β subunits. There are four collagen-binding integrins in mammalian cells: α1β1, α2β1, α10β1 and α11β1 [2–4]. The α subunit of these collagen receptors have an inserted (I) domain in their extracellular region that contains the collagen-binding site. The most widely distributed collagen-binding integrins are α1β1 (predominantly found in mesenchymal cells) and α2β1 (predominantly found in epithelial cells). α1β1 was expressed at the surface of mouse chondrocytes [5]. DDRs are receptors tyrosine kinases that recognize collagens as their ligands. DDRs constitute a unique subfamily of receptor tyrosine kinases containing a discoidin homology region in their extracellular domain [6]. There are two related DDRs: DDR1 and DDR2 are specifically expressed in epithelium and mesenchyme, respectively [6,7]. DDR2 was expressed and induced in the mouse osteoblasts and chondrocytes [8].

Both DDR and collagen-binding integrins bind a number of collagens with distinct specificity and recognize specific amino acid motifs within the collagen triple helix. α1β1 integrin is able to bind collagen types I, II, III, and V [3,9–11], and favor other subtypes such as type IV [10], while α2β1 integrin is the major receptor for type I collagen [9,11]. Currently, less is known about ligand binding preferences of the novel α10β1 and α11β1 integrins. DDR1 is primarily activated by type I–IV and VIII [12,13], whereas DDR2 responds to collagen I and to a lesser extent to type II, III, and V [12–14].

The interactions of collagens with integrin collagen receptors have been characterized, in particular for integrin α1β1 and α2β1. In addition to their importance as anchoring molecules, these heterodimeric receptors transmit bidirectional signals that regulate many important aspects of cell behavior including proliferation, differentiation, and survival [2,15,16]. Collagen-induced DDR signaling controls cell proliferation, adhesion and migration, as well as re-modeling of extracellular matrices [17–20]. For example, DDR receptors regulated the migration of primary human lung fibroblasts through collagen matrices and DDR2 was shown to be involved in fibroblast proliferation [21]. But very little is known about the collagen receptors and their expression, or the involvement during cell differentiation.

Interactions between proteins within the ECM and their receptors on the surface of mesenchymal cells might provide environmental signals that contribute to the progression of differentiation. Previous literature has shown that ECM proteins influence differentiation on hMSCs derived from bone marrow in in vitro cultures [22–25]. Type I collagen, the main ECM protein, was able to induce the osteoblastic differentiation of bone marrow cells, and furthermore promoted mineralization and up-regulated the osteogenic genes such as transcription factor Cbfa-1 and osteocalcin [26]. On the other hand, collagen receptors that bind to these ECM proteins promoted and inhibited differentiation of hMSCs. Previous studies showed that interactions of α2β1 integrin with collagen type I is required for osteoblastic differentiation of hMSCs [26,27] and the expression of osteogenic genes [28]. However, detailed studies of the expression of collagen receptors during the differentiation of hMSCs has not been described, particularly with regard to the interactions between collagen type I and collagen receptors during differentiation. The goal of the present study was to explore the expression of these collagen receptors during the chondrogenic and osteogenic differentiation of hMSCs, as well as to assess their roles in regulating collagen I-induced differentiation.

2. Materials and methods

2.1. Cell cultures

hMSCs were obtained from commercially available bone marrow aspirates from one male donor ≤25 years of age (Clonetics-Poietics, Walkersville, MD, USA) using methods previously reported [29]. Whole bone marrow aspirates were plated at 8–10 μl aspirate/cm² on 185 cm² tissue culture plates and cultivated until confluency (12–14 days) in 40 ml of ex vivo expansion medium consisting of Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin, 0.1 mm nonessential amino acids, and 1 ng/ml of basic fibroblast growth factor (bFGF) (Life Technologies, Rockville, MD, USA). hMSCs were maintained in a humidified tissue culture incubator at 37 °C with 5% CO₂. hMSCs were selected based on their ability to adhere to the tissue culture plastic; nonadherent hematopoietic cells were removed during medium replacement after approximately 5 days in culture. Medium was changed twice per week thereafter. hMSCs were subsequently detached using 0.25% trypsin/1 mM EDTA, replated at 5 × 10³ cells/cm², and cultured to 90% confluency to passage 1 cells.

2.2. Osteogenic and chondrogenic differentiation

Osteogenic differentiation medium included standard hMSC culture medium as listed above with the exception of 1 ng/mL bFGF. In addition, 0.05 mM L-ascorbic acid 2-phosphate (Sigma, USA), 1000 nM dexamethasone (Sigma, USA), 10 mM glycerol 2-phosphate disodium salt hydrate (Sigma, USA), and 100 ng/mL bone morphogenetic protein (BMP)-2 was added (Promocell, Germany). Passage 1 hMSCs were seeded at a density of 3 × 10⁴ cells/cm². To induce chondrogenic differentiation, cells were plated at a 1 × 10⁵ cells per well in a 96-well U-bottom suspension culture plate. Chondrogenesis was induced by culturing hMSCs in the presence of a defined hMSC medium that included 100 nM dexamethasone and 10 ng/ml transforming growth factor-beta(3) (TGF-beta(3)) (Promocell, Germany). Differentiation medium was changed two times per week.

2.3. Fluorescence-activated cell sorting (FACS)

FACS analysis was used to determine surface expression of DDR2 and integrin α11 in hMSCs. Membrane staining was performed with fluorescently labelled antibodies: anti-human DDR2 (R&D Systems (#290804); Abcam (ab98702), USA), and anti-human integrin α11 (Acris Antibodies Inc. (AP52249PU-N and R1255F), USA). Passage 1 cells were detached with 0.25% trypsin–EDTA, pelleted and resuspended in 300 μl DMEM. Cell suspensions were split into three aliquots; a control group without staining, anti-DDR2 or α11, and anti-human CD29 (known as integrin β1, Biolegend (303015), USA) conjugated with Alexa Fluor^® 488 as positive control. After addition of antibodies the samples were mixed and incubated for 30 min in the dark at 4 °C. The cells were washed with 1.5 ml DPBS and centrifuged for 5 min at 1500 rpm, 4 °C. The supernatant was removed, the cell pellet was suspended in 1 ml DPBS in polystyrene tubes (Falcon) and subjected to FACS analysis. Data was analyzed using FlowJo FACS analysis software 7.6 (Treestar Inc., USA).

2.4. mRNA expression analysis

Total RNA was extracted from the cells using an RNeasy Mini Kit (Qiagen, USA), following the supplier’s instructions. Extracted total RNA was used to synthesize cDNA using a High-Capacity cDNA Archive kit (Applied Biosystems, USA), following the supplier’s instructions. mRNA levels were determined either by quantitative real time PCR or by semi-quantitative reverse-transcriptase PCR (RT-PCR). Real time RT-PCR reactions were conducted and monitored using an Mx3000P (Stratagene^®). TaqMan Gene Expression System (Applied Biosystems, USA) with predesigned primer/probe sets were used to determine transcript levels of human genes DDR1, DDR2, integrin α1, α2, α10, α11 and β1 subunits, alkaline phosphatase (ALP), aggrecan and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The amplification program was as follows: 50 °C for 2 mins; 95 °C for 10 mins, followed by 40 amplification cycles of 95 °C for 15 seconds; and 60 °C for 1 min. Amplification specificity was validated by melting curve analysis. The mRNA expression changes (fold difference) were calculated after normalizing with GAPDH as an endogenous control. For RT-PCR, the number of amplification cycles was set at 25 to ensure that the amplifications were analyzed within an exponential phase. All PCR reactions from each cDNA sample were run in triplicates.

2.5. Western blots

Cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer (Invitrogen, USA). Protein concentrations were measured using the BCA protein assay (Thermo Fisher Scientific Inc., USA). The protein extract were resolved by 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Hybond-ECL nitrocellulose membranes (Invitrogen, USA). After blocking in Tris-buffered saline containing 5% BSA and 0.1% Tween-20, membranes were probed with primary antibodies against human DDR2 (R&D Systems (AF2538), USA) and integrin α11 (Acris Antibodies Inc. (AP52249PU-N), USA). Beta-actin was used as an internal positive control. For detection, the membranes were incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase and the blots were visualized with enhanced chemiluminescence.

2.6. Statistical analysis

All studies were performed in triplicate, and all experiments were repeated at least three times (n = 3). Results are presented as mean ± SD.

3. Results

3.1. Expression profile of collagen receptors in hMSCs

First, we investigated collagen receptor expression on both TCP and collagen type I substrates under undifferentiated conditions to profile the expression of collagen receptors in hMSCs. After 6 days of culture, mRNA expression was detected for collagen receptors DDR1, DDR2 and integrin subunits β1, α1, α2, α10 and α11. Integrin α11β1 and DDR2 showed a moderate to high level mRNA expression compared with the expression level of other collagen receptors, and both receptors had a higher mRNA level expression on collagen type I than on TCP. α1, α2, and α10 had a low expression. In contrast, the expression of DDR1 was not affected in hMSCs (Fig. 1).

Fig. 1.

hMSCs were cultured on TCP or TCP coated with type I collagen (Invitrogen, USA) for 6 days and mRNA was extracted to detect expression profiles of collagen receptors in hMSCs. RT-PCR was carried out with the indicated primers DDR1, DDR2, integrin subunits α1, α2, α10, α11 and β1 (A). The results are representative of mean fold increases ± S.D. of three independent experiments done in triplicate. For semi-quantitative RT-PCR, the number of amplification cycles was set at 25 to ensure that the amplification was analyzed within the exponential phase. PCR products were run on 2% agarose gels (B).

3.2. Expression level of DDR2 and integrin α11β1 in hMSCs by FACS

To test whether the expression level of DDR2 and integrin α11 subunit was expressed in hMSCs, we analyzed cell surface receptors level using FACS, and integrin β1 was used as positive control. As shown in Fig. 2, cell surface receptor expression levels of α11 was consistent with the mRNA expression level, while DDR2 showed a decrease when compared with the mRNA results.

Fig. 2.

hMSCs were collected after 6 day culture. The cells were incubated and stained with anti-DDR2, anti-integrin α11 and anti-integrin β1 antibodies. Flow cytometry was performed using FACSCalibur^{^TM} System (BD, USA). The frequency of positive results in the selected population is shown. DDR2 and α11 subunit showed 2.56% (A) and 8.72% (B) positive labeling, respectively, compared with negative and positive β1 subunit controls. The results are representative of mean ± S.D. of two independent experiments.

3.3. Activation dynamics of collagen receptors in differentiation of hMSCs

We further examined activation dynamics of collagen receptors during differentiation to see whether collagen receptor expression was altered by the differentiation commitment of hMSCs. As shown in Fig. 3, hMSC were harvested after culturing in osteogenic and chondrogenic medium for 6 days. The mRNA expression of collagen receptors DDR1, DDR2 and integrin subunits α1, α2, α10, α11 and β1 were examined separately by RT-PCR. Cells exhibited an osteoblastic phenotype at 6 days, which was determined by the increase in osteogenic marker expression. The chondrogenic commitment was assessed by chondrogenic marker expression. The expression of markers indicated early osteogenic and chondrogenic activities separately.

Fig. 3.

hMSCs were cultured on TCP or TCP coated with type I collagen in osteogenic and chondrogenic medium for 6 and 12 days and were harvested for total RNA isolation. RT-PCR was carried out with the indicated primers DDR1, DDR2, integrin α1, α2, α10, α11 and β1 subunits. The relative mRNA expression levels of collagen receptors was detected at different day points during osteogenic (A) and chondrogenic (B) differentiation. mRNA levels of ALP and aggrecan, osteogenic and chondrogenic markers, was detected at 0, 6 and 12 day (C). The results are representative of mean fold increases ± S.D. of three independent experiments done in triplicate.

mRNA expression of DDR2 and integrin α11β1 were peaked at day 6, followed by downregulation at 12 day in osteogenesis. Integrin subunit β1 showed a higher increase than α11 at 6 day, both subunits α11 and β1 decreased at 12 day. All collagen receptors showed higher mRNA expression when hMSCs were cultured on type I collagen substrate than on TCP during osteogenic differentiation. In chondrogenic differentiation, mRNA expression level of subunit α11 showed an increase at day 6 compared with the expression level of DDR2 that showed a decrease. α11 showed the same decrease as DDR2 at 12 days. α11 showed less mRNA expression on type I collagen substrate than on TCP at 6 and 12 days. The results suggested that integrin α11β1 was more active than DDR2 in chondrogenesis.

3.4. DDR2 and integrin α11 protein expression in differentiation of hMSCs

Western blots were carried out to measure DDR2 and α11 protein expression as indicated in Fig. 4. In osteogenesis, we focused on the role of DDR2 in osteogenic differentiation. DDR2 protein abundance showed an increase at day 6 and tended to decrease at 12 day with higher protein expression on type I collagen than on TCP, which was consistent with the changes in mRNA. These results suggest that the differentiation-dependent fluctuation of DDR2 expression level is a common feature of hMSCs. In chondrogenic differentiation, the α11 protein expression showed the same fluctuation tendency as the DDR2 expression, while α11 showed the same or a slight increase in protein expression on TCP than as on type I collagen during osteogenesis, which was different from the DDR2 protein expression during osteogenesis. These results suggested that the expression of integrin α11β1 was different from DDR2 by activation of type I collagen during chondrogenesis.

Fig. 4.

hMSCs were cultured on TCP or TCP coated with type I collagen in osteogenic and chondrogenic medium. Total protein was isolated after cells were lysed, and subjected to SDS-PAGE followed by anti-DDR2 and anti-α11 immunoblotting. Anti-β-actin was used as loading control. Protein expression of DDR2 during osteogenesis at different days and substrates was immunoblotted (A). The same commitment for integrin subunit α11 was performed in chondrogenesis (B). The results are representative of two independent experiments.

3.5. mRNA expression of DDR2 and integrin subunit β1 in differentiation of hMSCs

mRNA expression of both DDR2 and integrin subunit β1 increased at day 6 and tended to decrease at 12 day during osteogenic differentiations, and there was a higher expression level on type I collage than on TCP. The same association between DDR2 and subunit β1 was also found by activation of type I collagen during chondrogenesis, while this association was not found on TCP. These results suggested that mRNA expression of DDR2 was concomitant with the expression of subunit β1 by activation of type I collagen (Fig. 5).

Fig. 5.

mRNA expression level of DDR2 and integrin subunit β1 at 0, 6 and 12 days on type I collagen and TCP substrates during osteogenic (A) and chondrogenic (B) differentiation. The results are representative of two independent experiments.

4. Discussion

The expression and activation of two main collagen receptor subfamilies in hMSCs were initially profiled and DDR2 and integrin α11β1 were significantly altered compared with other receptors α1, α2, and α10. We further investigated the expression and activation of collagen receptors in hMSCs during osteogenic and chondrogenetic commitment. DDR2 and α11 were up-regulated during early stages of osteogenesis and chondrogenesis separately. The phenotypic similarities between DDR2- and collagen-deficient mice support in vitro evidence for DDR2 regulation of collagen synthesis in osteoblasts and chondrocytes [30–34]. A recent study showed that DDR2 played an important role in osteoblast differentiation and chondrocyte maturation via modulation of runx2 activation and also played an essential role in chondrocyte hypertrophy [8]. Notably, a recent report described a mutation in the human DDR2 gene that caused spondylometaepiphyseal dysplasia (SMED), a rare autosomal recessive disease [35].

We examined dynamic changes in DDR2 expression and collagen I-induced expression during osteogenic differentiation and found that DDR2 activation by type I collagen was paralleled by the changes in DDR2 expression on TCP. Maximal DDR2 expression was observed in mature osteoblasts at early stages of hMSC differentiation, suggesting that the differentiation-induced ECM microenvironment is required for full DDR2 expression. The unresponsiveness of hMSCs to DDDR2 upregulation under undifferentiated conditions is likely due to the lack of sufficient differentiation stimulation to DDR2. However, we did not find DDR2 upregulation of expression and activation during chondrogenic differentiation, unlike from previous research where DDR2 was active in both osteogenesis and chondrogenesis [8,35]. Thus these data suggest that DDR2 functions in osteogenic differentiation correlated with its activation by type I collagen.

Integrin α11β1 constitutes a recent addition to the integrin family [36]. α11 expression was present in various mesenchymal cells around cartilage anlage during human embryonic development, suggesting that α11 might be associated with the chondrogenic differentiation of mesenchymal cells [36]. We found upregulation expression of α11 during chondrogenic differentiation, and that its collagen I-induced expression was not paralleled by changes in α11 expression. Although both DDR2 and α11 showed a binding preference to type I collagen, they showed opposing expression by collagen I-induced expression during osteogenetic and chondrogenic differentiation, implying that differentiations has different effects on the expression and activation of DDR2 and α11 in hMSCs. The present results suggest that a11 plays a different role in chondrogenic differentiation unrelated to its activation by type I collagen.

The interplay between the DDRs and collagen-binding integrins has been observed in some cellular systems [37]. A recent study suggested that DDRs promoted α1β1- and α2β1- integrin mediated cell adhesion to collagen by enhancing integrin activation [37]. Interestingly, in our study, we found that the expression and collagen I-induced expression of DDR2 may be concomitant with another receptor integrin subunit β1 during osteogenetic differentiation. Little is known about DDR2 and β1 has been shown to be both required for cell adhesion on collagen in some cell types, and they may have mutual effects on the differentiation of mesenchymal cells.

5. Conclusion

In conclusion, the results presented in the present study suggest that DDR2 and integrin α11β1 play an important role in the differentiation of mesenchymal stem cells. DDR2 regulated the osteogenetic differentiation of hMSCS by activation by type I collagen, and for the first time we found that α11 played an important role in chondrogenic differentiation. DDR2 and integrin α11β1 functioned differently in osteogenesis and chondrogenesis. The data suggest that DDR2 was more specific in osteogenesis, while integrin a11b1 was more specific in chondrogenesis than in osteogenesis. DDR2 and α11 may play a role in the regulation of osteogenesis and chondrogenesis based on the differential expression of these receptors during lineage-dependent changes. The expression and activation of DDR2 may be concomitant with another receptor integrin subunit β1 during osteogenetic differentiation. Unraveling the importance of DDR2 and integrin α11β1-involved activity in mesenchymal cell differentiation represents an important direction related to control of stem cell fate and tissue regeneration.

Footnotes

Acknowledgements

The authors thank Bo An, Birgit Leitinge, Barbara Brodsky and David L. Kaplan for their contributions to this study.

Conflict of interest

None to report.

References

Hynes

R.O.

, Integrins: Bidirectional, allosteric signaling machines, Cell 110(6) (2002), 673–687.

Leitinger

and Hohenester

, Mammalian collagen receptors, Matrix Biol 26(3) (2007), 146–155.

Barczyk

Carracedo

and Gullberg

, Integrins, Cell Tissue Res 339(1) (2010), 269–280.

Pozzi

Wary

K.K.

Giancotti

F.G.

and Gardner

H.A.

, Integrin alpha1beta1 mediates a unique collagen-dependent proliferation pathway in vivo, J Cell Biol 142(2) (1998), 587–594.

Ronzière

M.C.

Aubert-Foucher

Gouttenoire

Bernaud

Herbage

and Mallein-Gerin

, Integrin alpha1beta1 mediates collagen induction of MMP-13 expression in MC615 chondrocytes, Biochim Biophys Acta 1746(1) (2005), 55–64.

Alves

Vogel

Mossie

Millauer

Höfler

and Ullrich

, Distinct structural characteristics of discoidin I subfamily receptor tyrosine kinases and complementary expression in human cancer, Oncogene 10(3) (1995), 609–618.

Labrador

J.P.

Azcoitia

Tuckermann

Lin

Olaso

Mañes

, The collagen receptor DDR2 regulates proliferation and its elimination leads to dwarfism, EMBO Rep 2(5) (2001), 446–452.

Zhang

Ren

Liu

, An essential role of discoidin domain receptor 2 (DDR2) in osteoblast differentiation and chondrocyte maturation via modulation of Runx2 activation, J Bone Miner Res 26(3) (2011), 604–617.

Nykvist

Ivaska

Käpylä

Pihlajaniemi

and Heino

, Distinct recognition of collagen subtypes by alpha(1)beta(1) and alpha(2)beta(1) integrins. Alpha(1)beta(1) mediates cell adhesion to type XIII collagen, J Biol Chem 275(11) (2000), 8255–8261.

10.

Kern

Eble

Golbik

and Kühn

, Interaction of type IV collagen with the isolated integrins alpha 1 beta 1 and alpha 2 beta 1, Eur J Biochem 215(1) (1993), 151–159.

11.

Käpylä

Ivaska

Riikonen

Nykvist

Pentikäinen

Johnson

, Integrin alpha(2)I domain recognizes type I and type IV collagens by different mechanisms, J Biol Chem 275(5) (2000), 3348–3354.

12.

Ruiz

P.A.

and Jarai

, Collagen I induces discoidin domain receptor (DDR) 1 expression through DDR2 and a JAK2-ERK1/2-mediated mechanism in primary human lung fibroblasts, J Biol Chem 286(15) (2011), 12912–12923.

13.

Vogel

Gish

G.D.

Alves

and Pawson

, The discoidin domain receptor tyrosine kinases are activated by collagen, Mol Cell 1(1) (1997), 13–23.

14.

Shrivastava

Radziejewski

Campbell

Kovac

McGlynn

Ryan

T.E.

, An orphan receptor tyrosine kinase family whose members serve as nonintegrin collagen receptors, Mol Cell 1(1) (1997), 25–34.

15.

Miranti

C.K.

and Brugge

J.S.

, Sensing the environment: A historical perspective on integrin signal transduction, Nat Cell Biol 4(4) (2002), E83–E90.

16.

Berrier

A.L.

and Yamada

K.M.

, Cell-matrix adhesion, J Cell Physiol 213(3) (2007), 565–573.

17.

Vogel

W.F.

Abdulhussein

and Ford

C.E.

, Sensing extracellular matrix: An update on discoidin domain receptor function, Cell Signal 18(8) (2006), 1108–1116.

18.

Ali

B.R.

Akawi

N.A.

John

Karuvantevida

N.S.

Langer

, Trafficking defects and loss of ligand binding are the underlying causes of all reported DDR2 missense mutations found in SMED-SL patients, Hum Mol Genet 19(11) (2010), 2239–2250.

19.

Gross

Beirowski

Harvey

S.J.

McFadden

Chen

Tam

, DDR1-deficient mice show localized subepithelial GBM thickening with focal loss of slit diaphragms and proteinuria, Kidney Int 66(1) (2004), 102–111.

20.

Bargal

Cormier-Daire

Ben-Neriah

Le Merrer

Sosna

Melki

, Mutations in DDR2 gene cause SMED with short limbs and abnormal calcifications, Am J Hum Genet 84(1) (2009), 80–84.

21.

Ruiz

P.A.

and Jarai

, Discoidin domain receptors regulate the migration of primary human lung fibroblasts through collagen matrices, Fibrogenesis Tissue Repair 5(3) (2012). doi:10.1186/1755-1536-5-3.

22.

Santiago

J.A.

Pogemiller

and Ogle

B.M.

, Heterogeneous differentiation of human mesenchymal stem cells in response to extended culture in extracellular matrices, Tissue Eng Part A 15(12) (2009), 3911–3922.

23.

Kishimoto

Hattori

Nakamura

Amano

Kanatani

Tanaka

, Expansion and characterization of human bone marrow-derived mesenchymal stem cells cultured on fragmin/protamine microparticle-coated matrix with fibroblast growth factor-2 in low serum medium, Tissue Eng Part C Methods 15(3) (2009), 523–527.

24.

Kundu

A.K.

and Putnam

A.J.

, Vitronectin and collagen I differentially regulate osteogenesis in mesenchymal stem cells, Biochem Biophys Res Commun 347(1) (2006), 347–357.

25.

Dieudonne

F.X.

Sévère

Biosse-Duplan

Weng

J.J.

and Marie

P.J.

, Promotion of osteoblast differentiation in mesenchymal cells through Cbl-mediated control of STAT5 activity, Stem Cells 31(7) (2013), 1340–1349.

26.

Mizuno

and Kuboki

, Osteoblast-related gene expression of bone marrow cells during the osteoblastic differentiation induced by type I collagen, J Biochem 129(1) (2001), 133–138.

27.

Takeuchi

Suzawa

Kikuchi

Nishida

Fujita

and Matsumoto

, Differentiation and transforming growth factor-beta receptor down-regulation by collagen-alpha2beta1 integrin interaction is mediated by focal adhesion kinase and its downstream signals in murine osteoblastic cells, J Biol Chem 272(46) (1997), 29309–29316.

28.

Xiao

Wang

Benson

M.D.

Karsenty

and Franceschi

R.T.

, Role of the alpha2-integrin in osteoblast-specific gene expression and activation of the Osf2 transcription factor, J Biol Chem 273(49) (1998), 32988–32994.

29.

Altman

G.H.

Horan

R.L.

Martin

Farhadi

Stark

P.R.

Volloch

, Cell differentiation by mechanical stress, FASEB J 16(2) (2002), 270–272.

30.

Kano

Marín de Evsikova

Young

Wnek

Maddatu

T.P.

Nishina

P.M.

, A novel dwarfism with gonadal dysfunction due to loss-of-function allele of the collagen receptor gene, Ddr2, in the mouse, Mol Endocrinol 22(8) (2008), 1866–1880.

31.

Labrador

J.P.

Azcoitia

Tuckermann

Lin

Olaso

Mañes

, The collagen receptor DDR2 regulates proliferation and its elimination leads to dwarfism, EMBO Rep 2(5) (2001), 446–452.

32.

Stacey

Bateman

Choi

Mascara

Cole

and Jaenisch

, Perinatal lethal osteogenesis imperfecta in transgenic mice bearing an engineered mutant pro-alpha 1(I) collagen gene, Nature 332(6160) (1988), 131–136.

33.

Pereira

Khillan

J.S.

Helminen

H.J.

Hume

E.L.

and Prockop

D.J.

, Transgenic mice expressing a partially deleted gene for type I procollagen (COL1A1). A breeding line with a phenotype of spontaneous fractures and decreased bone collagen and mineral, J Clin Invest 91(2) (1993), 709–716.

34.

Helminen

H.J.

Kiraly

Pelttari

Tammi

M.I.

Vandenberg

Pereira

, An inbred line of transgenic mice expressing an internally deleted gene for type II procollagen (COL2A1). Young mice have a variable phenotype of a chondrodysplasia and older mice have osteoarthritic changes in joints, J Clin Invest 92(2) (1993), 582–595.

35.

Bargal

Cormier-Daire

Ben-Neriah

Le Merrer

Sosna

Melki

, Mutations in DDR2 gene cause SMED with short limbs and abnormal calcifications, Am J Hum Genet 84(1) (2009), 80–84.

36.

Tiger

C.F.

Fougerousse

Grundström

Velling

and Gullberg

, alpha11beta1 integrin is a receptor for interstitial collagens involved in cell migration and collagen reorganization on mesenchymal nonmuscle cells, Dev Biol 237(1) (2001), 116–129.

37.

Bihan

Chang

Huang

P.H.

Farndale

R.W.

and Leitinger

, Discoidin domain receptors promote alpha1beta1 and alpha2beta1-integrin mediated cell adhesion to collagen by enhancing integrin activation, PLoS One 7(12) (2012), e52209. doi:10.1371/journal.pone.0052209.