Molecular Biology of the Cell
Vol. 16, 881–890, February 2005
Laminin-5 Induces Osteogenic Gene Expression in Human
Mesenchymal Stem Cells through an ERK-dependent
Robert F. Klees,* Roman M. Salasznyk,* Karl Kingsley,†William A. Williams,*
Adele Boskey,‡and George E. Plopper*§
*Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180-3596;‡Hospital for Special Surgery,
New York, NY 10021; and†University of Nevada, Las Vegas, School of Dental Medicine, Las Vegas, NV
Submitted August 12, 2004; Accepted November 16, 2004
Monitoring Editor: Mark Ginsberg
The laminin family of proteins is critical for managing a variety of cellular activities including migration, adhesion, and
differentiation. In bone, the roles of laminins in controlling osteogenic differentiation of human mesenchymal stem cells
(hMSC) are unknown. We report here that laminin-5 is found in bone and expressed by hMSC. hMSC isolated from bone
synthesize laminin-5 and adhere to exogenous laminin-5 through ?3?1 integrin. Adhesion to laminin-5 activates extra-
cellular signal-related kinase (ERK) within 30 min and leads to phosphorylation of the osteogenic transcription factor
Runx2/CBFA-1 within 8 d. Cells plated on laminin-5 for 16 d express increased levels of osteogenic marker genes, and
those plated for 21 d deposit a mineralized matrix, indicative of osteogenic differentiation. Addition of the ERK inhibitor
PD98059 mitigates these effects. We conclude that contact with laminin-5 is sufficient to activate ERK and to stimulate
osteogenic differentiation in hMSC.
Human mesenchymal stem cells (hMSC) are multipotent
cells found within the bone marrow and periosteum (Barry
and Murphy, 2004). Typically they differentiate into chon-
drogenic, adipogenic, or osteogenic lineages, but recent ev-
idence suggests that hMSC can also express phenotypic
characteristics of endothelial, neural, smooth muscle, skele-
tal myoblast, and cardiac myocyte cells (Pittenger and Mar-
tin, 2004). The mechanisms governing hMSC differentiation
are not well understood, but the ability of these cells to self
renew and develop into numerous tissues makes their po-
tential use in clinical applications quite promising.
Extracellular matrix (ECM) proteins are well-known reg-
ulators of multiple cellular functions, including differentia-
tion. The laminin (Ln) family of ECM proteins are ubiqui-
tously expressed but are especially abundant in the
basement membrane of many epithelial and endothelial tis-
sues, where they mediate cell attachment, migration, and
tissue organization in conjunction with other ECM proteins
(Malinda and Kleinman, 1996). Each laminin molecule is a
heterotrimer, composed of an ?-, ?-, and ?- subunit. The
subunits share homology with one another and upon com-
bining through disulfide bonds form an asymmetric cross-
like structure with one long and three short arms (Colognato
and Yurchenco, 2000). The Ln-5 isoform is composed of ?3,
?3, and ?2 subunits. Expression of the ?2 subunit has only
been found in Ln-5, whereas the ?3 subunit is found in both
Ln-6 and Ln-7. Ln-5 is bound by ?2?1, ?3?1, ?6?1, and ?6?4
integrin receptors (Decline and Rousselle, 2001), all of which
are found in hMSC (Pittenger et al., 1999). The role of Ln
family members in osteogenic differentiation is not known
(Roche et al., 1999), though expression of the ?2 chain has
been previously detected in bone marrow (Siler et al., 2002).
Ln-5 is expressed in distinct temporal and spatial patterns
in developing epithelial tissues and influences tissue com-
partmentalization and cellular phenotypes from early em-
bryonic development onward (Aberdam et al., 1994; Timpl
and Brown, 1996). These tissues include the oral, nasal, and
olfactory epithelium, intestine and stomach, bronchi and
bronchioli, and breast (Virtanen et al., 1995; Virtanen et al.,
1996; Orian-Rousseau et al., 1996; Stahl et al., 1997; Thorup et
al., 1997). In these tissues Ln-5 has been shown to promote
cellular growth, morphogenesis, and wound healing.
Ln-5 is typically found in tissues derived from ectoderm
and endoderm. We recently discovered the expression of
Ln-5 in the intimal layer of the vasculature, where it is
expressed by vascular smooth muscle cells and plays a role
in controlling the growth and migration of these cells in
response to peptide growth factors (Kingsley et al., 2001,
2002a, 2002b). The fact that we found this Ln isoform in a
mesodermally derived tissue implies that Ln-5 may play a
role in regulating the growth and differentiation of other
mesodermal tissues as well, including bone.
Article published online ahead of print in MBC in Press on Decem-
ber 1, 2004 (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.
§Corresponding author. E-mail address: firstname.lastname@example.org.
Abbreviations used: BMP, bone morphogenic protein; COLL-I, col-
lagen-I; ELF, enzyme-linked fluorescence; ERK, extracellular-related
kinase; FN, fibronectin; FTIR, Fourier transform infrared; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; hMSC, human mesen-
chymal stem cells; Ln, Laminin; MEK1, MAPK kinase; nd-blotto, 5%
non-dairy creamer in PBS ? 0.2% Tween 20; OS, osteogenic supple-
ment; pERK, phosphorylated ERK; pRunx2, phospho-runx2/
CBFA-1; RIPA, radioimmunoprecipitation assay; VN, vitronectin.
© 2005 by The American Society for Cell Biology881
Recently it has been discovered that the ECM proteins
collagen-I (COLL-I) and vitronectin (VN), found in bone,
induce osteogenic differentiation comparable to that ob-
served with soluble osteogenic supplements (Salasznyk et
al., 2004). This suggests that other bone ECM proteins, and
their associated integrin receptors, may also play a similar
role in the differentiation of hMSC. Integrins stimulate in-
tracellular signaling pathways via short cytoplasmic tails
that act as docking sites for interactions with downstream
signaling molecules (Schwartz, 2001). There are at least six
classes of signaling molecules stimulated by integrin activa-
tion: protein tyrosine kinases, serine/threonine kinases,
lipid kinases, lipid phosphatases, protein phosphatases, and
ion fluxes (Schwartz and Ginsberg, 2002). The roles that
these molecules play in the differentiation of hMSC are for
the most part unknown.
One of the potential signal transduction pathways that
might direct the differentiation of hMSC is the mitogen-
activated protein (MAP) kinase pathway. Extracellular sig-
nal-related kinase (ERK) is a member of the MAP kinase
family that stimulates the differentiation of hMSC into
osteoblasts via phosphorylation of the osteogenic transcrip-
tion factor runx2/CBFA-1 (Jaiswal et al., 2000). However, the
cellular pathways linking ECM contact with ERK activation
in hMSC are unknown. The goal of this study was to exam-
ine the role of Ln-5-associated ERK signaling in osteogenic
differentiation of hMSC. We hypothesized that Ln-5 stimu-
lates osteogenic differentiation of hMSC by ?3?1 integrin-
mediated activation of ERK, with subsequent activation of
runx2/CBFA-1 and expression of osteogenic genes.
MATERIALS AND METHODS
Tissue culture media (DMEM) and penicillin G-streptomycin sulfate (GPS)
were purchased from Mediatech (Cellgro, Herndon, VA). Fetal bovine serum
(FBS) was purchased from Gemini Bio-Products (Woodland, CA). Trypsin-
EDTA and purified bovine COLL-I were obtained from Sigma Chemical Co.
(St. Louis, MO). Laminin-1 G4 domain was cloned into a pET vector, ex-
pressed in BL21DE3 bacteria, and purified using a nickel column. Purified
mouse collagen IV and Ln-1 were purchased from Collaborative Research
(Bedford, MA) Purified laminin-5 was generously provided by Desmos (San
Diego, CA). Purified human plasma VN and human plasma fibronectin (FN)
were purchased from Chemicon International (Temecula, CA). The ?v?3
(catalogue no. MAB1976) and the ?1–6 and ?1–4 integrin function-blocking
antibodies (Alpha integrin blocking and IHC kit, catalogue no. ECM 430; Beta
integrin screening kit, catalogue no. ECM 440) were purchased from Chemi-
con International (Temecula, CA). Rabbit polyclonal IgG Anti-g-actin (cata-
logue no. AAM01-A) antibody was from Cytoskeleton (Denver, CO). Rabbit
polyclonal IgG antibodies against anti-ERK1/2 (catalogue no. AB3053), os-
teopontin (catalogue no. AB1870), osteocalcin (catalogue no. AB1857), and
phosphoserine (catalogue no. AB1603) were obtained from Chemicon Inter-
national. Mouse monoclonal IgG antibodies for ?6integrin (catalogue no.
CBL458), ?3integrin (catalogue no. MAB2056), and Ln-5 (catalogue no.
MAB1947) were obtained from Chemicon International. Rabbit polyclonal
IgG phosphospecific antibodies against anti-ERK 1/2 (pTpY185/187) (cata-
logue no. 44–680) was from Biosource International (Camarillo, CA). Mouse
monoclonal IgG antibodies against anti-Runx2/Cbfa1 were purchased from
MBL International (Watertown, MA). Rabbit polyclonal IgG antibody for Ln-5
was provided by Vito Quaranta (Vanderbilt University, Nashville, TN).
Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and HRP-
conjugated goat anti-rabbit IgG secondary antibodies were obtained from
Jackson ImmunoResearch (West Grove, PA). Protein A/G-agarose beads and
the MEK1 inhibitor PD98059 were purchased from Calbiochem (San Diego,
CA). RT-PCR primers listed in Table 1 were purchased from IDT Technolo-
gies (Coralville, Iowa). The protein assay kit was purchased from Pierce
(Rockford, IL). Unless otherwise specified, the other standard reagents were
obtained from Fisher Scientific (Fair Lawn, NJ).
Cryopreserved hMSC were purchased from Cambrex (Walkersville, MD) and
were grown according to the manufacturer’s instructions. Briefly, cells were
plated at 5 ? 103cells/cm2in a T75 flask (75 cm2) for continuous passaging
in DMEM medium supplemented with 10% FBS, 1% l-glutamine (29.2 mg/
ml), penicillin G (10,000 U/ml) and streptomycin sulfate (10,000 ?g/ml).
Medium was changed twice weekly and cells were detached by trypsin-
EDTA and passaged into fresh culture flasks at a ratio of 1:3 upon reaching
confluence. Cultures were incubated at 37°C in a humidified atmosphere
containing 95% air and 5% CO2.
Laminin-5 matrix was isolated from rat bladder carcinoma 804G cells as
described previously (Plopper et al., 1996)
For in vitro osteogenic assays, hMSC were passaged three times before they
were inducted and plated at densities of 3.1 ? 103cells/cm2in 0.2 ml/cm2of
medium on 100-mm Falcon dishes (78.5 cm2). The following day (day 0), we
replaced the culture medium with fresh control medium in the absence or
presence of osteogenic supplements (OS) containing 0.1 ?M dexamethasone,
0.05 mM ascorbic acid-2-phosphate, and 10 mM ?-glycerophosphate (Cam-
brex). In each experiment, control and OS media–treated cells were processed
in parallel. For inhibition of MEK1, PD98059 in dimethyl sulfoxide (DMSO)
was added to control and OS media at a final concentration of 50 ?M twice a
week during media changes unless otherwise specified. The final concentra-
tion of DMSO never exceeded 0.1%, and the same amount of DMSO vehicle
Table 1. RT-PCR primers
Gene namePrimer sequences
ALPL (alkaline phosphatase)Forward 5?-GGGGGTGGCCGGAAATACAT-3?
SSP1 (osteopontin) 564
CBFA1 (core binding factor alpha 1)421
GAPDH (Glyceraldehyde-3-phosphate dehydrogenase)1000
Laminin alpha 1 chain780
Laminin beta 1 chain 659
Laminin-5 alpha 3 chain269
Laminin-5 beta 3 chain374
Laminin-5 gamma 2 chain436
R. F. Klees et al.
Molecular Biology of the Cell882
was added to control conditions. For all other assays, suspensions of cells
were incubated with PD98059 (50 ?M) 15 min before plating. At days 8, 16,
and 21, cultures were assayed as described below.
Immunological Detection of Ln-5 in Bone
Timed pregnancy rats were handled according to the University of Nevada,
Las Vegas Protocol for Animal Care and Use, Protocol Number R701-1297-
136: Developmental studies in rats and mice. Twelve-, 14-, 16-, and 18-d
timed-pregnancy embryos were harvested from pregnant rats under general
anesthesia and the mothers were killed using intracardial injection of Nem-
butal (sodium pentabarbital) ?100 mg/kg.
Embryos were fixed at 4°C for 24 h in each of three successive paraformal-
dehyde solutions (4% wt/vol) containing 5, 10, and 15% sucrose, respectively.
Embryos were then halved using standard dissection tools and frozen in
Miles Tissue-TEK OCT 4583 embedding medium (Naperville, IL) in standard
cryomolds using liquid freon. Tissue sections (8 ?m thick) were cut using a
Microm Cyostat HM 505E (Microm GmbH, Walldorf, Germany) and adhered
to poly-l-lysine (0.8 mg/ml)-coated glass slides for photobleaching and
Tissues were maintained at 27°C in a tinoxid-filter greenhouse at constant
oxygen (20%) and CO2(5%) to remove autofluoresence. Tissues were then
sectioned using the cryotome and stained suing the Molecular Probes (Eu-
gene, OR) Enzyme-Linked-Fluorescence ELF-97 Immunohistochemistry Pro-
tocol, MP 06600 using TR1 or CM6 monoclonal antibody (mAb), for the
gamma and alpha chains of laminin-5, respectively (Plopper et al., 1996a).
Stained tissues were visualized using a Leica DM LB immunofluorescence
microscope (Deerfield, IL) with DAPI and FITC filter sets. ELF-substrate
excitation and emission wavelengths are 360 and 535 ? 18 nm, respectively,
and images were collected using SPOT diagnostic digital imaging equipment.
Mineralized Bone: Fourier Transform Infrared Analysis
The presence of apatite in cell matrix was detected by Fourier transform
infrared (FTIR) analysis of ground powders. Cell layers, collected in 50 mM
ammonium bicarbonate (pH 8.0) after 21 d, were lyophilized and analyzed as
potassium bromide (KBr) pellets on a Bio-Rad FTS 40-A spectrometer (Bio-
Rad Microscience, Cambridge, MA). The data were collected under nitrogen
purge, and the spectral baseline was corrected and analyzed using GRAMS/
386 software (Galactic Industries, Salem, NH) as previously described (Kato et
al., 2001). The mineral content was calculated based on the spectrally derived
mineral-to-matrix ratio (the integrated areas of the phosphate absorbance
[900–1200 cm?1] and protein amide I band [1585–1720 cm?1]).
Immunoprecipitation of Runx2 and Western Blotting
Whole cell extracts were prepared by harvesting overnight serum-deprived
cells or cells after 8 and 16 d in culture (DMEM ? 0.1% FBS) with ice-cold
radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris,
1% Triton X-100, 0.3 mM sodium vanadate, 1% deoxycholic acid, 0.2% SDS,
pH 7.4). For immunoprecipitation, samples were precleaned twice with 20 ?l
of protein A/G-agarose beads for 30 min followed by pelleting of beads.
Mouse monoclonal anti-Runx2, 4 ?l, was added and incubated for 2 h at 4°C
with shaking. The immune complexes were collected on the addition of 20 ?l
of protein A/G-agarose beads and subsequently incubated for 1 h at 4°C,
followed by centrifugation at 12,000 ? g for 10 min. Precipitates were washed
thoroughly and suspended three times in ice-cold RIPA buffer, centrifuged at
2500 ? g for 10 s, and resuspended in Laemmli sample buffer. Proteins were
denatured at 100°C for 5 min, resolved by 8% SDS-PAGE, and electrophoreti-
cally transblotted to Trans-Blot nitrocellulose membranes (0.2 ?m; Bio-Rad
Laboratories, Hercules, CA). The membranes were incubated with blocking
solution (5% nonfat dried milk in 1? phosphate-buffered saline [PBS] ? 0.2%
Tween-20 [PBST]) for 1 h and then probed with various primary antibodies
(1:500) overnight at 4°C. After three washes with PBST, membranes were
incubated with HRP-conjugated secondary IgG (1:25,000) for 1 h, followed by
another three washes with PBST. Immunoreactive bands were detected using
the SuperSignal Chemiluminescent reagent (Pierce) and quantitatively ana-
lyzed by normalizing band intensities to the controls on scanned films by
IMAGEJ software (National Institutes of Health, Bethesda, MD).
Cell adhesion assays were performed as previously described using Sarstedt
96-well suspension cell culture plates (Newton, NC; Plopper et al., 1998).
Tissue culture plates were coated with purified ECM proteins at a concentra-
tion of 20 ?g/ml for 1 h at room temperature. Wells were washed twice with
PBS and incubated with nd-blotto (5% nondairy creamer in PBS ? 0.2%
Tween 20) for 30 min before the assay. Cells were allowed to attach for 30 min
at 37°C and were subsequently fixed with 3% paraformaldehyde, washed
twice in PBS, and incubated in crystal violet dye for 15 min. Wells were
washed thoroughly with water and the violet dye was extracted with 10%
SDS solution. Absorbance was measured using a Tecan SPECTAFluor spec-
trophotometer (Hillsborough, NC) at 595 nm, and relative adhesion was
compared with cells attached to nd-blotto.
Integrin-blocking adhesion assays were performed according to the proce-
dure above, but the cells were incubated with a functional integrin-blocking
antibody for 30 min at 37°C, with vortexing every 5 min before plating.
Cell migration assays were performed using 8-?m MIC plates from Millipore
(Danvers, MA). Filters were coated with purified ECM proteins (COLL-I, VN,
Ln-5, or FN) at a concentration of 20 ?g/ml or nd-blotto for 1 h at room
temperature before assay. Basal chambers for the nd-blotto wells were filled
with migration medium (DMEM ? 1% sodium pyruvate ? 1? GPS), whereas
the basal chambers for the ECM containing wells were filled with control
medium. Cell suspensions in migration medium were seeded at a density of
5 ? 103cells per well. Migrations were allowed to run for 18 h at 37°C. Filters
were then incubated for 30 min with 5 ?M calcein AM (Molecular Probes,
Eugene, OR) and washed thoroughly with PBS. Residual cells were swabbed
from the top of the wells to avoid false readings. To quantitate migration,
plates were read at 485Ex/535Em with a Tecan SPECTAFluor spectropho-
tometer. Relative fluorescence values for each experimental condition were
expressed relative to FN and nd-blotto controls.
RNA was isolated from 10 ? 108hMSC cultured for 16 d in the presence or
absence of PD98059 (50 ?M) on tissue culture plastic (?OS media) or on Ln-5
in control media. Total RNA was isolated using the RNeasy mini kit (Qiagen,
Valencia, CA). RT-PCR was performed with the OneStep RT-PCR Kit (Qia-
gen) and a 96-well thermal cycler (MJ Research, Waltham, MA) using the
primers listed in Table 1, which were designed by the Lasergene v5.0 program
(DNASTAR, Madison, WI). One microgram of template RNA was used per
reaction. The reverse transcription step ran for 30 min at 50°C, followed by
PCR activation for 15 min at 95°C. Thirty amplification cycles were run,
consisting of 1-min denaturation at 94°C, 1 min of annealing at 58°C, and 1
min of extension at 72°C. Final extension was allowed to run 10 min at 72°C.
Reaction products were separated by gel electrophoresis using a 1% agarose
gel. Bands were visualized by UV illumination of ethidium-bromide–stained
gels and captured using a ChemiImager 4400 Gel imaging system (Alpha
Innotech, San Leandro, CA). Band intensity was quantitatively analyzed by
IMAGEJ software for each gene and was normalized to corresponding glyc-
eraldehyde-3-phosphate dehydrogenase (GAPDH) values.
hMSC were grown on glass coverslips for 16 d, fixed with acetone, and
blocked with PBS/1% bovine serum albumin (BSA) for 30 min. Primary
mouse antibodies for Ln-5, ?3integrin, and ?6integrin were diluted 1:200 in
1? PBS ? 1% BSA and added to cells for 1 h. Double staining was done with
a rabbit polyclonal for Ln-5 with either of the integrin antibodies. FITC and
TRITC secondary antibodies were added for 1 h and cover slips were
mounted using Prolong antifade medium (Molecular Probes). Cells were
visualized with a Nikon TE2000-S inverted fluorescence/phase contrast mi-
croscope (Garden City, NY) equipped with a digital SPOT camera.
Cultured monolayers of hMSC cultured for 16 days were washed twice with
PBS and extracted with 0.5 N HCl, and the resulting lysate was shaken for 5 h
at 4°C, followed by centrifugation at 2000 ? g for 10 min. The supernatant was
utilized for calcium determination, according to the manufacturer’s instruc-
tions contained in Sigma Kit 587. Total calcium was calculated from absor-
bance at 575 nm using standard solutions prepared in parallel as calibration
All experiments were repeated a minimum of two times, and the represen-
tative data were presented as mean ? SE. Statistical analyses were preformed
using Student’s unpaired t test, and a p value ? 0.05 was considered signif-
Laminin-5 Is Expressed in Bone and hMSC
We utilized enzyme-linked fluorescence (ELF)-based immu-
nohistochemistry and RT-PCR analysis to detect Ln-5 in
adult bone tissue and hMSC. Positive staining for Ln-5 ?3
and ?2 chains were detected in rat rib periosteum (Figure 1,
E and F, respectively), whereas no visible staining was ob-
served in the negative controls (Figure 1, A–D). Similar
staining was seen in all adult and embryonic bone tissues
Laminin-5 and Osteogenesis of hMSC
Vol. 16, February 2005883
Because mesenchymal stem cells are found in the perios-
teum (Barry and Murphy, 2004), we hypothesized that they
were the source of the Ln-5 in bone. After routine fixation
and Triton X-100 permeabilization of cells plated for 16 d on
glass coverslips, staining for Ln-5 appeared to be largely
intracellular, in the endoplasmic reticulum and Golgi com-
plex (Figure 2B). Intense staining was also visible for the ?3
integrin as punctate dots after 16 d (Figure 2A), confirming
that hMSC express this integrin. Both the ?3 integrin and
Ln-5 were detected in double-labeled cells (Figure 2, C and
D). Ln-5 was also colocalized with the ?6 integrin but with
weaker intensity for the ?6 integrin (unpublished data).
Extraction of hMSC with 20 mM HN4OH, which solubilizes
virtually all cellular proteins but leaves behind the insoluble
ECM (Gospodarowicz, 1984), removed all evidence of Ln-5
staining (unpublished data). No staining was observed in
the negative control (unpublished data). RT-PCR confirmed
the presence of the three Ln-5 chains in hMSC cultured for
16 d on tissue culture plastic (Figure 2E). Ln-1 chains, ?1 and
?1, were also detected as a positive control. Adhesion and
migration of hMSC was greater on Ln-5 than with other
ECM proteins tested, including the G4 domain of Ln-1 in
adhesion assays (Figure 3). In 30-min adhesion assays, inte-
grin chain-specific blocking antibodies against ?3 and ?1
integrin subunits reduced adhesion to Ln-5 by ?50 and 60%,
respectively, whereas antibodies against ?6 and ?4 reduced
adhesion by 20–25% (Figure 4). Control antibodies targeting
integrin subunits that do not bind Ln-5 (?1, ?2, ?v) did not
ERK as a Mediator of Osteogenic Differentiation in hMSC
Plated on Ln-5
ERK is a common component of many integrin signaling
pathways. To test our hypothesis that adhesion to Ln-5
induces osteogenic differentiation of hMSC via ERK sig-
naling, we plated hMSC on Ln-5 and observed the levels
of phosphorylated ERK (pERK). Cells plated on Ln-5 for
30 min contained four and six times more phosphorylated
ERK 1 and ERK 2, respectively, than cells plated on poly-
l-lysine–coated control surfaces (Figure 5). Cells plated
on tissue culture plastic and treated with osteogenic sup-
plement (OS) media also had more phosphorylated ERK 1
and ERK 2 than that of controls. Cells kept in suspension
exhibited no detectable pERK1 and only a trace amount of
pERK2. Addition of the MAP Kinase Kinase 1 (MEK1)
inhibitor PD98059, which blocks ERK activation, elimi-
units to the periosteum of rat rib. Enzyme-
linked fluorescence technology was used to
detect immunostaining (arrows) of Ln ?3 and
?2 chains with CM6 and TR1 antibodies in
frozen sections of adult rat rib (E and F, re-
cence (A), reduced autofluorescence follow-
ing exposure to UV light (B, bone-cartilage
junction; and D, rib synovium), background
staining from uv-treated section stained with
normal mouse IgG (C). All images taken at
10? magnification. Cortical bone shattered
during frozen sectioning.
Immunolocalization of Ln-5 sub-
on glass coverslips for 8 d and then were fixed and stained for ?3
integrin (A and C) and human Ln-5 (B and D). (A) Magnification,
40?. (B–D) Magnification, 10?. Images in C and D were taken of the
same microscopic field. (E) RT-PCR was used to amplify the indi-
cated mRNA transcripts from hMSC grown on tissue culture plastic.
The ?1 and ?1 chains, found in Ln-1, were included as a positive
control. GAPDH was amplified as a positive control.
hMSC express Ln-5 in culture. (A–D) Cells were grown
R. F. Klees et al.
Molecular Biology of the Cell 884
nated pERK1 and drastically reduced the amount of
pERK2 detected in both the Ln-5– and OS-treated cul-
Activation of the transcription factor Runx2 was detected
by immunoprecipitation and immunoblotting for phospho-
serine residues. Cells grown on Ln-5 for 8 d exhibited a
threefold increase in phospho-runx2/CBFA-1 (pRunx2)
compared with control cells plated on tissue culture plastic.
Cells treated with OS media exhibited a 2.5-fold increase in
pRunx2 over the same time course (Figure 6). Addition of
PD98059 reduced the amount of detectable pRunx2 by
?50% in OS and Ln-5 conditions, even as early as 1 d after
To measure the effects of Ln-5 plating on hMSC osteogenic
differentiation, we performed RT-PCR for three osteogenic
marker genes (osteopontin, osteocalcin, and alkaline phos-
phatase [ALP]), which are downstream targets of Runx2, as
well for Runx2 itself (Figure 7). We found that adhesion to
Ln-5 for 16 d activated expression of all three marker genes,
greater than that found in hMSC grown in OS media over
the same time course. Cells plated on poly-l-lysine control
expressed only trace amounts of each marker gene. Addition
of PD98059 to the experimental conditions reduced the ex-
pression of all three marker genes to levels below or equal to
that of the control. Runx2 levels were comparable across all
controls and conditions, regardless of the presence of the
To confirm the RT-PCR data, Western blots were per-
formed for osteopontin and osteocalcin in cultured hMSC
(Figure 8). Control cultures expressed no detectable levels of
these proteins in the presence or absence of PD98059. How-
ever, both proteins were detected in OS media and Ln-5
samples, and the expression of these proteins was greatly
diminished when PD98059 was added to the cultures.
The presence of hydroxyapatite in the cell matrix is a
reliable sign of osteogenic differentiation. Hydroxyapatite
formation was measured using FTIR analysis of insoluble
matrix washed with ammonium bicarbonate. The results,
expressed as the mineral:matrix ratio of secreted ECM, were
2.0 and 2.8 for cultures grown for 21 d on poly-l-lysine and
Ln-5, respectively (Figure 9). The very broad band in the nu4
region (500–650 cm?1) is indicative of very poorly crystal-
line apatite or of apatite of very small crystal size. Fully
differentiated adult osteoblasts produce a matrix that yields
a ratio of 5.4 over the same time course (Salasznyk et al.,
2004). Thus, cells grown on Ln-5 contain more hydroxy-
apatite than cells grown on tissue culture plastic, even after
only 21 d.
Fibronectin is found in the periosteum (Nilsson et al.,
1998) and supports hMSC adhesion (Figure 3A), but is
thought to play a role in adipogenic differentiation rather
than osteogenic differentiation of stromal cells (Urs et al.,
2004). Adhesion to fibronectin for 30 or 60 min induced
phosphorylation of ERK 2 (42 kDa), but not ERK 1 (44 kDa;
assay of hMSC adhesion to purified ECM proteins. Adherent cells
were stained with crystal violet and then solubilized in SDS, and
absorbance was determined at 595 nm. Values represent mean ? SD
(n ? 5). (B) Cells were plated in the upper chamber of a MIC filter
migration plate (Millipore) and allowed to migrate toward the
lower chamber through a 8-?m filter coated with Ln-5. Controls had
filters coated with COLL-I, VN, FN, or nd-blotto. Each condition
was repeated in 12 wells.
hMSC bind to and migrate on Ln-5. (A) Static 30-min
as per Figure 3A were performed in the presence of integrin block-
hMSC use ?3?1 integrin to bind Ln-5. Adhesion assays
Laminin-5 and Osteogenesis of hMSC
Vol. 16, February 2005885
Figure 10A). Similarly, plating cells on fibronectin failed to
induce calcium deposition in hMSC cultured for 16 d (Figure
10B). Thus, fibronectin is not an osteogenic stimulus of
hMSC under our culture conditions.
Ln-5 is typically found in tissues derived from ectoderm and
endoderm, such as skin, gut, lung, and other epithelia
(Yamamoto et al., 2001). Our data presented here, combined
with our recent discovery of Ln-5 in vascular smooth muscle
cells (Kingsley et al., 2001, 2002a, 2002b), suggests that Ln-5
is also expressed in mesoderm-derived tissues as well. Why
has Ln-5 gone undetected in these tissues previously? We
believe we were able to uncover its presence in rat perios-
teum because of our high level of detection sensitivity. ELF
(enzyme-labeled fluorescence) is a powerful tool in immu-
nohistochemistry because it provides significant signal am-
plification over conventional indirect immunofluorescence
techniques (Paragas et al., 2002). In addition, we developed a
method for significantly reducing autofluorescence in meso-
dermal tissues (Kingsley et al., 2001). This has allowed us to
generate a very high signal-to-noise ratio, allowing the de-
tection of both the ? and ? chains of Ln-5, even in tissues
where it is expressed in relatively low levels.
Our immunolocalization data raise an important issue
regarding ECM synthesis in hMSC. That Ln-5 is expressed in
low abundance in situ (Figure 1) and is not incorporated into
an insoluble matrix in hMSC cultured on 2D surfaces (Figure
2) suggests that hMSC may not assemble their own ECM.
This is somewhat surprising for most cell types, but virtually
all studies involving ECM effects on 2D cultures of hMSC
utilize exogenous ECM proteins rather than hMSC matrix. A
recent study demonstrated that these cells synthesize ECM
proteins when grown in 2D, but that the proteins are local-
ized to the cytoplasm; only when the cells are cultured in a
3D environment are the proteins deposited as a matrix
(Grayson et al., 2004).
Our findings are also consistent with what is known about
Ln-5 assembly. Ln-5 assembly into epithelial basement
membranes requires direct cell contact (Plopper et al., 1996b)
and requires the formation of disulfide bonds (Falk-Marzil-
lier et al., 1998). It is quite possible that hMSC lack the
machinery to perform this assembly.
2D tandem mass spectrometry (120 ?g total protein, de-
tection threshold ?200 pmol) of whole-cell hMSC lysate
grown in 2D fails to identify any ECM proteins (Salasznyk,
Westcott, Klees, Ward, Xiang, Vandenberg, Bennet, Plopper,
unpublished results). Likewise, mass spectrometry of the
NH4OH-insoluble protein from 60 ? 106cultured hMSC
fails to detect any ECM proteins (Klees, unpublished data).
This leads us to conclude that, although it is clear that hMSC
synthesize the mRNA for the Ln-5 constituent chains and
produce immunologically detectable levels of the ?2 chain in
Ln-5, hMSC are not likely responsible for assembling this
Ln-5 into bone matrix. Instead, this function may be pro-
vided during development by chondrocytes and in adults by
committed osteoblasts. Osteoblasts colocalize with hMSC in
the periosteum and bone marrow and are well known to
synthesize and assemble both collagenous and noncollag-
enous elements of bone matrix (Marks and Odgren, 2002).
hMSC. (A) Cells kept in suspension (susp.), or
plated for 30 min on poly-l-lysine (Poly-L-L), tis-
sue culture plastic with OS media (?OS), or on
Ln-5 were probed by immunoblot for phosphory-
lated ERK (top row), total ERK (middle row), or
actin as a loading control (bottom row). Where
indicated by a “?” symbol, 50 ?M PD98059 was
added to the culture conditions. (B and C) Densi-
tometric measurements of band intensities for
phospho-ERK 1 and phospho-ERK 2, respectively.
Adhesion to Ln-5 activates ERK in
R. F. Klees et al.
Molecular Biology of the Cell886
Mesenchymal stem cells have been found in many loca-
tions, including the bone marrow, trabecular bone, adipose
tissue, skeletal muscle, and periosteum (Barry and Murphy,
2004). Our detection of Ln-5 in the periosteum suggests the
possibility that hMSC may be the source of this protein. Ln-5
has been shown to support the adhesion of primary osteo-
progenitor cells (Roche et al., 1999). Our RT-PCR and immu-
nohistochemistry data demonstrate that Ln-5 is expressed
by hMSC in culture, supporting our supposition. The RT-
PCR data also confirm that hMSC express the ?1 and ?1
chains of Ln-1, which are also found in the periosteum
(Nilsson et al., 1998) but which do not support adhesion or
osteogenic differentiation of hMSC (Salasznyk et al., 2004).
Expression of the ?3 laminin chain also indicates that Lami-
nin-6 and Laminin-7 may be present in hMSC, because they
share this ? chain with Ln-5 (Champliaud et al., 1996). To
date, these isoforms have not been reported in bone.
In 30-min adhesion assays, hMSC interact with Ln-5
through known Ln-5 receptors, primarily ?3?1. All four
Ln-5 integrin receptors (?2?1, ?3?1, ?6?1, ?6?4) are ex-
pressed by hMSC (Pittenger et al., 1999) and may be utilized
at different times during hMSC maturation. For example,
Nguyen et al. (2000) have suggested that in human foreskin
keratinocytes, these receptors function cooperatively to me-
diate cellular response to Ln-5 binding. Specifically, they
suggest that initial adhesion to Ln-5 is mediated by ?3?1,
and later, more stable contact is established through ?6?4
integrins, which form hemidesmosomes. During wound re-
pair, this process is reversed: cells switch from a RhoGTPase-
dependent adhesion via ?6?4 integrin to a PI3K-dependent
adhesion and spreading via ?3?1 integrin (Nguyen et al.,
2000). Our data suggest that stem cells establish early con-
tact with Ln-5 through ?3?1; it is not yet known what role
the other integrins may play in hMSC contact with Ln-5.
In addition to engaging at least three different integrin
receptors, Ln-5 is also known to promote several integrin-
associated signaling pathways. Our data demonstrate that
adhesion to Ln-5 stimulates ERK 1 and ERK 2, primarily
through ?3?1 integrin. ?3?1 is thought to be the primary
receptor for initial adhesion of most epithelial cells to Ln-5,
and this binding affects the ability of other receptors to bind
Ln-5 at later times (Belkin and Stepp, 2000). Members of the
transmembrane 4 superfamily of proteins (tetraspanins) are
known to associate tightly with ?3?1 integrin and form a
link between the receptor and protein kinase C (PKC; Zhang
et al., 2001). PKC activity is dependent on the intracellular
calcium concentration in developing osteoblasts (Matsu-
moto, 1995). Tetraspanins can also modulate other integrin
signaling and may function in reorganization of the actin
cytoskeleton (Berditchevski and Odintsova, 1999). We have
noted a significant rearrangement of F-actin in differentiat-
ing hMSC (Salasznyk et al., 2004). Integrins also stimulate
ERK activity (Carloni et al., 2004), which we hypothesize is
crucial for osteogenic differentiation. In breast cells, Ln-5/
?6?4 integrin complexes are believed to be conduits for
signals from the extracellular environment to intracellular
signaling pathways including MAP kinase (Giancotti, 1996;
Gonzales et al., 1999). Collectively, these data suggest that
hMSC use pathways similar to those found in other cells to
transduce Ln-5 binding into intracellular signaling activity.
How, then, does hMSC adhesion to Ln-5 result in osteo-
genic gene expression? Osteoblast differentiation from bone
marrow progenitor cells has been described as a series of up
to seven steps, each defined by a change in gene expression
(Aubin, 1998). More recent studies suggest that these steps
are a continuum, rather than distinct events (Ryoo et al.,
1997; Hou et al., 1999; Smith et al., 2000). This in turn explains
why gene expression patterns are somewhat heterogeneous,
making true osteoblast differentiation patterns difficult to
define. The most critical of these events is the activation/
phosphorylation of the master bone gene runx2 (Lian et al.,
2004). Runx2 is responsible for expression of osteogenic
marker genes, including osteopontin, osteocalcin, and ALP.
Runx2/CBFA-1 is a substrate of ERK, which can be activated
by soluble osteogenic supplements (Bruder et al., 1997;
Jaiswal et al., 2000).
Though it is expressed in relatively low abundance, Ln-5
influences responsiveness to other extracellular stimuli in
vascular smooth muscle (Kingsley et al., 2002a, 2002b). Os-
teoprogenitor cells adhere better to laminins than to other
ECM proteins (Roche et al., 1999). What is clear is that Ln-5
plays more than a simple adhesive role during hMSC dif-
ferentiation: FN supports adhesion and migration of these
cells but does not induce expression of an osteoblastic phe-
notype (Salasznyk et al., 2004). Although adhesion to Ln-5
does induce ERK activation, which is common with adhe-
sion to many ECM proteins, this binding also induces ex-
pression of osteogenic genes, which is observed upon bind-
ing to only a subset of bone ECM proteins (COLL-I, VN).
Our finding that fibronectin fails to activate ERK 1 and does
not induce calcium deposition suggests that ERK 1–specific
signals may be necessary for osteogenic differentiation. That
this differentiation occurs in the absence of any other extra-
cellular stimulus strongly suggests that, although ERK 1
activation is involved, additional signaling pathways likely
ylation. Cells were plated for 1 d (top row) or 8 d (middle and
bottom rows) on tissue culture plastic in the absence (CTL) or
presence of OS media (?OS), or on Ln-5, and then lysed, and
runx2/CBFA-1 was immunoprecipitated. The immunoprecipitated
proteins were separated by SDS-PAGE and probed by immunoblot
for phosphoserine (top two rows) or total runx2/CBFA-1 as a load-
ing control (bottom row). Where indicated by a “?” symbol, 50 ?M
PD98059 was added to the culture conditions. (B and C) Densito-
metric measurements of bands for pRunx2 on days 1 and 8, respec-
Adhesion to Ln-5 stimulates runx2/CBFA-1 phosphor-
Laminin-5 and Osteogenesis of hMSC
Vol. 16, February 2005887
play a role in controlling osteogenic gene expression. In fact,
ERK is known to control activation of SMADs and the AP-1
transcription complex, both of which play a role in regulat-
ing osteogenic gene expression (Franceschi, 1999; Franceschi
and Xiao, 2003).
Initiation of these signaling changes must begin with an
external stimulus. In addition to stimulating integrin recep-
tors, Ln-5 may act as a repository for soluble osteogenic
factors. It is well known that the bone ECM is a repository
for bone morphogenic proteins (BMPs), which induce dif-
ferentiation of osteoprogenitor cells through regulation of
osteoblast-specific transcription factors. BMPs bind to chor-
din like cysteine-rich repeats found in several ECM proteins
(O’leary et al., 2004). Both the ?2 and ?3 chains of Ln-5
contain potential cysteine-rich regions that may bind BMPs.
Other growth factors may interact with Ln-5 and/or its
associated proteins within the bone matrix.
To our knowledge, this report is the first demonstration
that Ln-5 controls differentiation of a mesodermally derived
tissue. Our data suggest a model whereby adhesion to Ln-5
via ?3?1 integrin stimulates ERK activation. This in turn
leads to phosphorylation of runx2/CBFA-1 and subsequent
expression of osteopontin, osteocalcin, and ALP. The fact
that Runx2/CBFA-1 activation and osteogenic gene expres-
sion are sensitive to PD98059 supports our model that ad-
hesion to Ln-5 alone, in the absence of any exogenous solu-
ble stimulants, issufficient
differentiation and that this is mediated by ERK 1. We have
observed significant increases in matrix mineralization in
cells plated on Ln-5 for only 21 d, though achieving miner-
alization comparable to that found with fully mature osteo-
blasts will likely take much longer. By necessity, we have
focused on the earlier, inductive stages of osteogenic differ-
entiation in this study.
in hMSC. (A) RT-PCR was performed on hMSC grown for 16 d on
tissue culture plastic in the absence (CTL) or presence of OS media
(?OS), or on Ln-5. Where indicated by a “?” symbol, 50 ?M
PD98059 was added to the culture conditions. Indicated genes were
amplified using primers listed in Table 1. GAPDH was amplified as
a loading control. (B–E) Densitometric analysis of bands in A for (B)
ALP, (C) osteocalcin, (D) osteopontin, and (E) runx2/CBFA-1.
Ln-5 induces expression of osteopontin and osteocalcin
Cells were plated for 16 d under the same conditions as in Figure 7 and
probed by immunoblot for osteocalcin (OC) and osteopontin (OP).
hMSC plated on Ln-5 express osteocalcin and osteopontin.
hMSC plated on tissue culture plastic (A) or Ln-5 (B) for 21 d. The
mineral:matrix is computed by comparing the area of the phosphate
peak (mineral) to the amide peak (protein) shown.
Ln-5 induces matrix mineralization. FTIR analysis of
R. F. Klees et al.
Molecular Biology of the Cell888
No in vitro culture conditions can recreate the complex
microenvironment and intracellular communication neces-
sary for successful osteogenesis (Fox et al., 2000). Successful
bone formation is a highly complicated process that clearly
requires careful integration of signals from multiple stimuli,
including many ECM proteins. Our data demonstrate that
Ln-5 is capable of inducing osteogenic gene expression and
matrix mineralization in vitro, but do not suggest that Ln-5
alone is the osteogenic stimulus for hMSC, nor that Ln-5 is
necessary for bone formation. Ln-5 and the other noncollag-
enous molecules found in bone collectively make up no
more than 5% of bone protein, and it is quite likely that
many of these 5% are functionally redundant with regard to
assisting in bone formation and maintenance.
This work was supported by Public Health Service Grant 5R01EB002197-02
from the National Institute of Biomedical Imaging and Bioengineering
(NIBIB; to G.E.P.) and by SEED funds from Rensselaer Polytechnic Institute.
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