Identification of Fibronectin IIICS Variants in Human Bone Marrow Stroma

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 α₄β₁ and α₅β₁ integrins. The α₄β₁ 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 α₄β₁ 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 (Table1). The importance of the IIICS domain in hematopoiesis lies in its possession of two α₄β₁ 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 α₄β₁ is present in the adjacent heparin-binding domain, Hep II.5 As the interaction between α₄β₁ 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 α₄β₁ 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) (Fig1A). 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 pCR^TM 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.

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 α₄β₁integrin 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.