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Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 410-411
CORRESPONDENCE
Identification of Fibronectin IIICS Variants in Human Bone Marrow
Stroma
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LETTER |
To the Editor:
The distribution of fibronectin in the bone marrow stromal
extracellular matrix will influence adhesion and localization of hematopoietic stem and progenitor cells that bind to its two major cell-binding domains via  4 1 and
51 integrins. The
41 integrin may have a role in adhesion
of more primitive hematopoietic progenitor cells; both human long-term
culture-initiating cells and colony-forming unit (CFU)-mix progenitors
adhere to the fibronectin COOH-terminal heparin-binding domain through
41 integrin1 and primitive murine CFU-spleen (CFU-S) day 12 colony-forming cells adhere to the
CS1-containing fibronectin fragment.2 Important
differential splicing occurs at the type-III connecting segment (IIICS)
in human fibronectin that codes for five potential RNA variants (Table 1). The importance of the IIICS domain in
hematopoiesis lies in its possession of two
41 integrin binding sites, represented by
synthetic peptides called CS1 and CS5, which lie in separate, independently spliced regions so that either one, both, or neither site
may be expressed.3,4 A third weaker site recognized by
41 is present in the adjacent
heparin-binding domain, Hep II.5 As the interaction between
41 integrin and fibronectin will depend
on the products of alternative splicing of the IIICS domain, eg, only
two of the possible five products contain the CS1 sequence that binds
41 with highest affinity, we set out to
determine expression of the IIICS variants at the RNA level in marrow
stromal cells grown in long-term culture using reverse transcriptase-polymerase chain reaction (RT-PCR). The identity of the
resulting PCR products was confirmed by restriction enzyme digestion
and DNA sequencing. Normal marrow was cultured in standard long-term
culture conditions to produce a confluent stromal layer. Three days
before harvesting, the stromas were irradiated with 1,500 cGy of gamma
radiation from a 137Caesium source to destroy hematopoietic
cells. Total RNA was extracted from the adherent stromal cell layer and
mRNA isolated. First-strand cDNA was generated by reverse transcription
using avian RT primed with oligo-dT and the cDNA was
amplified by primers complementary to sequences on each side of the
IIICS region (5 -GAATAATCAGAAGAGCGAGCC and 3-ACTCAGAAGTGTCCTGGAATG).
Four distinct bands were seen consistent with expression of variants
V120 (expected band size = 464 bp), V89 or V95 (expected band
sizes = 371 or 389, respectively), V64 (expected band size = 296),
and V0 (expected band size = 104) (Fig
1A). The control with no RT confirmed that
the products originated from expressed stromal RNA. The PCR products
were cloned using the TA cloning system (Invitrogen, San Diego, CA).
Seven PCR inserts were all of one size consistent with either V89 or
V95, two were the size expected for V64, and one insert corresponded to
the largest V120 variant. PCR inserts from selected clones were
identified by restriction enzyme analysis with BglII, which
cuts the IIICS-A sequence (containing CS1) but not the IIICS-B or
IIICS-C sequences. BglII also cuts the pCRTM II
vector resulting in two bands of sizes 1,155 bp and 3,148 bp with the
IIICS-A/IIICS-B sequence (V89 variant) (as shown in Fig 1B) lanes 2 and
4. Lane 3 contains the IIICS-B sequence alone (V64 variant). None of
the clones contained the V95 variant. Sequencing the inserts using the
original IIICS primers confirmed the identity of V89 and V64. The
identity of the largest and smallest variants (V120 and V0) was not
confirmed by restriction enzyme analysis or sequencing, but they were
clearly present in the original RT-PCR (Fig 1A). A summary of the
variants detected in stroma is shown in Table 1.
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| Fig 1.
(A) RT-PCR amplification of marrow stromal RNA using
primers complementary to sequences on each side of the alternatively
spliced IIICS region of fibronectin. Ten microliters of PCR products
was run out on a TBE/2% agarose gel. Lane 1, reaction
with RT; lane 2, control without reverse transcriptase; lane 3, DNA
marker. The four bands in lane 1 are consistent with variants V120 (464 bp), V95 or 89 (389 or 371 bp), V64 (296 bp), and V0 (104 bp). (B)
Restriction enzyme digests of selected clones prepared from TA cloning
RT-PCR products of IIICS region of fibronectin. Three microliters of
digested products was run on a TBE/1% agarose gel.
BglII cuts the IIICS region when the CS1 sequence is
present. A second BglII site in the pCRTM II
vector results in 1,155- and 3,148-bp products in the presence of a
CS1/IIICS-B insert (V89) as shown in lanes 2 and 4. The band in lane 3 is consistent with a IIICS-B insert (V64) within the linearized vector
(4,124 bp).
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A previous study has described differences between IIICS variants in a
variety of human normal and tumor cell types, though not marrow
stroma.6 Although the CS1 sequence is present in a murine
bone marrow-derived stromal cell line,2 we show here for
the first time that normal human bone marrow stroma grown in long-term
culture expresses four of the IIICS variants: V120, V89, V64, and V0.
The V95 variant was not present. Expression of two CS1-containing
variants together with two variants that do not have this sequence
provides a mechanism for controlling 41
integrin receptor-mediated interactions of hematopoietic progenitor
cells at the level of mRNA splicing. It is also probable that protein
modification in situ, which may change domain structures, is another
potential level of control. Finally, the precise localization of the
variant proteins within the marrow extracellular matrix would allow for fine control of progenitor and stem cell interactions with resulting changes in adhesion, migration, and growth.
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ACKNOWLEDGEMENT |
K.P.S. is a clinical research fellow of the Cancer Research
Campaign.
Karen P. Schofield
Departments of Medical
Oncology and Experimental Haematology
Paterson Institute for Cancer
Research
Manchester, UK
Martin J. Humphries
Wellcome
Trust Centre for Cell-Matrix Research
University of Manchester
Manchester, UK
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REFERENCES |
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Humphries MJ, Komoriya A, Akiyama SK, Olden K, Yamada KM:
Identification of two distinct regions of the type III connecting segment of human plasma fibronectin that promote cell type-specific adhesion.
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4.
Mould PA, Komoriya A, Yamada KM, Humphries MJ:
The CS5 peptide is a second site in the IIICS region of fibronectin recognised by the integrin 41.
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Identification of a novel recognition sequence for the integrin 41 in the COOH-terminal heparin-binding domain of fibronectin.
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Cell-type-specific expression of alternatively spliced human fibronectin IIICS mRNAs.
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10:662, 1990[Abstract/Free Full Text]
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