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(Circulation. 1997;96:1729-1732.)
© 1997 American Heart Association, Inc.

Articles

Bmx Tyrosine Kinase Is Specifically Expressed in the Endocardium and the Endothelium of Large Arteries

Niklas Ekman, BSci; Athina Lymboussaki, MSci; Imre Västrik, PhD; Krista Sarvas, BMed; Arja Kaipainen, MD, PhD; ; Kari Alitalo, MD, PhD

From the Molecular/Cancer Biology Laboratory, Haartman Institute, University of Helsinki (Finland).

Correspondence to Dr Kari Alitalo, Molecular/Cancer Biology Laboratory, Haartman Institute, PL 21 (Haartmaninkatu 3), 00014 University of Helsinki, Helsinki, Finland. E-mail Kari.Alitalo{at}Helsinki.FI

	Abstract

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Background The growth and differentiation of endothelial cells are regulated by signal transduction through tyrosine protein kinases. Recently, a novel cytoplasmic tyrosine kinase gene, Bmx (Bone Marrow tyrosine kinase gene in chromosome X), was identified in human bone marrow RNA and found to be expressed predominantly in myeloid hematopoietic cell lineages. Our preliminary analyses indicated that the Bmx gene was also highly expressed in human heart.

Methods and Results Mouse Bmx cDNA was isolated, sequenced, and found to encode a polypeptide 91% identical to the human Bmx tyrosine kinase. Northern blotting and in situ hybridization of tissue sections indicated that Bmx mRNA is specifically expressed in the endocardium of the developing heart as well as in the endocardium of the left ventricle and in the endothelium of large arteries in adult mice. A weak signal was seen also in coronary arterial endothelium.

Conclusions Bmx shows a unique specificity of expression among tyrosine kinase genes and may be involved in signal transduction in endocardial and arterial endothelial cells. The results suggest that specific signal transduction mechanisms are present in such endothelia.

Key Words: genes • signal transduction • endocardium • endothelium

	Introduction

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Tyrosine protein kinases (TKs) are essential components of signal transduction in endothelial cells. Several of the TKs function as transmembrane receptors, transducing growth factor signals to the cytoplasm.¹ The extracellular domains of the receptor TKs are responsible for ligand binding, while the intracellular TK domains transmit the activation signals through phosphorylation of cellular polypeptides. Five different endothelial cell receptor TKs are known and are encoded by two different gene families.¹ So far, nonreceptor tyrosine kinases relatively specific for endothelial cells have not been reported.

The Bmx TK belongs to the so-called Btk subfamily.² The four proteins encoded by members of this gene family share substantial homology, including typical SH2 and SH3 domains upstream of the TK domain. Special features of these TKs include a so-called pleckstrin homology (PH) domain in the N-terminal region³ and expression in various hematopoietic cells. The Tec TK is expressed in all murine hematopoietic cell lines examined.⁴ Itk⁵ and Btk⁶ are selectively expressed at certain stages of lymphocyte development, and the expression of the Txk TK has been assigned to T cells.⁷

The Bmx gene was isolated during screening for novel TK genes expressed in human bone marrow.⁸ Because the gene was mapped to chromosome Xp22.2, it was called Bmx, Bone Marrow tyrosine kinase gene in chromosome X. In comparison with other Btk family members, Bmx lacks the so-called P-X-P motifs but has extra peptides in between the PH and SH3 and SH2 domains. The SH3 sequence does not conform precisely to the described consensus. Here we have isolated mouse Bmx cDNAs and studied Bmx expression in tissues by Northern blotting and in situ hybridization.

	Methods

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Isolation and Analysis of Mouse Bmx cDNA Clones
Approximately 1x10⁶ bacteriophage lambda clones from a 12-day post coitum (p.c.) mouse embryo cDNA library (Novagen) were screened with a radiolabeled BamHI fragment (nucleotides 192 to 1831) of human Bmx cDNA (sequence accession number X83107). One positive clone containing 1.7 kb of the open reading frame and 3' untranslated sequence as well as a polyA sequence was isolated and subcloned as three fragments, which were sequenced from both strands. The remaining 5' portion of the cDNA was obtained by isolation of the first Bmx coding exon from a mouse genomic DNA library in the lambda FIX II vector (Stratagene), with the use of a PCR fragment containing human Bmx nucleotides 23 to 162 as a probe. Primers were designed on the basis of the obtained sequence for PCR amplification of the remaining part of mouse cDNA with the use of mouse heart Quick-Clone cDNA (Clontech) as the template. The PCR reaction conditions were denaturation at 94°C for 60 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 30 seconds for 30 cycles in a reaction volume of 50 µL. The PCR fragment obtained was subcloned into the pCRII vector (Clontech) and sequenced. Two independent amplifications and clonings were carried out from the same cDNA with the use of Dynazyme polymerase (Finnzymes).

Analysis of mRNA Expression in Tissues
A Northern blot containing 2 µg of polyadenylated RNAs from various mouse tissues (Clontech) was hybridized with the 1.0-kb Bmx cDNA fragment probe and washed under stringent conditions, according to the manufacturer's instructions.

The mouse Bmx antisense and sense RNA probes were synthesized from linearized pBluescript II SK+ plasmid (Stratagene; La Jolla, Calif), containing a HindIII-EcoRI fragment from mouse Bmx cDNA (nucleotides 1302 to 2369, Genbank accession number AF012104), by incorporation of [³⁵S]-UTP using T7 and T3 polymerases after linearization with EcoRI and HindIII, respectively. In situ hybridization of paraffin sections was performed as previously described.⁹

	Results

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Cloning of Mouse Bmx cDNA
The mouse Bmx cDNA structure and the isolated clones are shown schematically in Fig 1A. The location of the different protein domains encoded by the cDNA as well as translational start and stop codons and polyadenylation signal are marked in the figure. A comparison of the deduced amino acid sequences of mouse and human Bmx genes is shown in Fig 1B. Comparison of the degree of sequence identity with other members of the Btk/Emt/Tec/Txk/Bmx TK family allowed an unequivocal identification of the clone as the homologue of human Bmx (data not shown).

View larger version (76K): [in this window] [in a new window]   Figure 1. Structure of the mouse Bmx cDNAs and comparison of the deduced amino acid sequences of mouse and human Bmx. A, Structure of the Bmx tyrosine kinase cDNA. The regions encoding the different protein homology domains have been marked as PH, pleckstrin homology domain; SH3, deleted Src homology domain 3; SH2, Src homology domain 2; and TK, tyrosine kinase domain. The translational start and stop codons ATG and TGA as well as the polyadenylation sequence (pA) also are marked. The cDNA subclones and the genomic clone (G) are indicated. The PCR primers used to amplify the 5' third of the cDNA (1: 5'-ACGACAATATGGAGAGTA-3' 2: 5'-ATCGAGATTTTCCTCTTC-3') are indicated with arrows. E indicates EcoRI site; H, HindIII site. B, Predicted amino acid sequence of mouse Bmx cDNA compared with the human sequence. Gaps have been introduced for optimal alignment. The PH domain, the SH2 domain, and the SH3 domain are boxed. The TK domain is marked with arrows, the ATP binding region is indicated with a horizontal line, and the autophosphorylation residue (Y) is boxed. The two tandemly repeated peptide sequences in human Bmx are indicated with horizontal arrows above the sequence. Note that most of the second repeat including the N-terminal part of the SH3-like domain is missing from mouse Bmx. C, Northern blotting and hybridization analysis of polyA+ RNA isolated from the indicated mouse tissues, with mouse Bmx cDNA as the probe.

Localization of Bmx mRNA in Tissues
When mouse Bmx cDNA was used to probe a Northern blot containing polyA+ RNA from various adult mouse tissues, clear-cut mRNA signals were obtained only from the heart and lung, whereas the other tested tissues showed very weak signals (Fig 1C).

Mouse embryos and adult tissues were processed for in situ hybridization with mouse Bmx cDNA as the probe. Sections from 8.5-day and 9.5-day p.c. embryos were negative for Bmx mRNA. In 10.5-day (data not shown) and 12.5-day p.c. embryos, the Bmx autoradiographic signals decorated the heart endocardium (Fig 2). Both ventricular and atrial endocardium were positive. In addition, the endothelium of the dorsal aorta, the subclavian arteries, and the intervertebral arteries showed a strong hybridization signal, whereas the cardinal vein was negative (Fig 3, A and B). Also in other areas, the signal seemed to be restricted to arterial endothelium. In the caudal part of 14.5-day p.c. mouse embryos, the hybridization signal was detected in the umbilical arteries (Fig 3, C and D). In embryos of 16.5-day p.c., the aortic endothelial signals were weaker (Fig 3, E and F). The Bmx sense probe did not give a signal in any of these sections (see Fig 3, G and H).

View larger version (83K): [in this window] [in a new window]   Figure 2. Expression of Bmx mRNA in 12.5-day mouse embryos. A and B show results of hybridization with antisense and sense probes, respectively. Note that Bmx mRNA is localized in the embryonic endocardium both in the atria and in the ventricles (arrows). In addition, the aortic endothelial (AE) cells give a Bmx mRNA signal, which can be discerned from the reflection of light from hematopoietic cells within the aorta (see B). The cardinal vein (CV) is negative for Bmx mRNA expression. Bar=250 µm.

View larger version (151K): [in this window] [in a new window]   Figure 3. Expression of Bmx in embryonic and adult tissues. A and B, Transverse section of the upper thoracic region from a 12.5-day embryo via dark field and light field photomicrography. The endothelium of the aortic arch (arrow) as well as the endothelium of the common carotid artery is positive. In the dorsal part, the vertebral arteries (arrowheads) also show a signal. C and D, Transverse section of a 14.5-day embryo, in which the umbilical arteries are strongly Bmx mRNA positive. E, F, G, and H, Hybridization of the aorta of a 16.5-day embryo with the antisense and sense probes, respectively. J through M, Sections of adult heart. J and K, Bmx signal decorates the heart endocardium (vertical arrows) and parts of the endocardium of the proximal aorta (bent arrows) but not the endothelium of the heart valves. L and M, Endocardial signal in the upper left corner (vertical arrow) and a weak Bmx signal in the coronary artery (filled arrowhead) but not in the coronary vein (empty arrowhead). Note the blood clots within the vessel lumen, giving false-positive signals (asterisk). Bar=100 µm.

In adult mice, autoradiographic signals were obtained from the heart endocardium of the left ventricle and from the aorta (Fig 3, J and K). Interestingly, the endocardium of the right ventricle was negative (data not shown), whereas coronary arteries showed a weak hybridization signal (Fig 3, L and M). Signals could not be detected from the lung or kidney.

	Discussion

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Our data suggest for the first time a function for the Bmx TK, a member of the Btk/Emt/Tec/Txk/Bmx TK family, outside the hematopoietic system. We have previously shown that the Bmx gene is expressed in bone marrow cells, in CD34+ hematopoietic cells from umbilical cord blood, and in peripheral blood granulocytes.^{8 10} Although the Tec TK is expressed in hepatocytes and hepatomas,⁴ previous studies have indicated that the Btk/Emt/Tec/Txk/Bmx TKs function mainly in certain lineages of hematopoietic cells, where they are activated by several upstream signal transducers.² In addition, recent data indicate that the PH domain of Btk interacts with specific phospholipids,¹¹ and such binding may be modulated by lipid kinases and phosphatases activated during receptor-mediated signal transduction in these cells. The upstream signals activating the Bmx TK are as yet unknown, but our experiments have indicated that one of the downstream components of the Bmx signal transduction pathway is the Stat transcription factor (P. Saharinen, N. Ekman, K. Sarvas, P. Parker, K. Alitalo, and O. Silvennoinen; unpublished data).

The expression of the Bmx gene apparently begins around day 9.5 to day 10.5 and then continues throughout further development. By Northern hybridization, Bmx transcripts were identified in adult mouse heart and lung. On the basis of the present in situ hybridization results, the mRNA signal in the lung sample is probably derived from the large arteries present in this material. The signal in the heart sample was considered to be derived from the adult endocardium. Interestingly, the coronary arteries also showed a weak but definitive Bmx mRNA signal. An interesting possibility, raised by the present findings, is that Bmx is involved in relaying blood flow–related or blood pressure–related signals in endocardial and arterial endothelial cells. Such signals could then lead to a long-term readjustment of gene expression in the affected cells.

	Acknowledgments

 
This study was supported by the Finnish Cancer Organizations, the Finnish Academy, the Sigrid Juselius Foundation, the University of Helsinki, and the State Technology Development Centre. We thank Mari Helanterä and Tapio Tainola for excellent technical assistance.

Received May 22, 1997; revision received July 14, 1997; accepted July 21, 1997.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Mustonen T, Alitalo K. Endothelial receptor tyrosine kinases involved in angiogenesis. J Cell Biol. 1995;129:895-898.[Free Full Text]

2. Vihinen M, Smith E. Structural aspects of signal transduction in B-cells. Immunology. 1996;16:251-274.

3. Musacchio A, Gibson T, Rice P, Thompson J, Saraste M. The PH domain: a common piece in the structural patchwork of signalling proteins. Trends Biochem Sci. 1993;18:343-348.[Medline] [Order article via Infotrieve]

4. Mano H, Ishikawa F, Nishida J, Hirai H, Takaku F. A novel protein-tyrosine kinase, tec, is preferentially expressed in liver. Oncogene. 1990;5:1781-1786.[Medline] [Order article via Infotrieve]

5. Gibson S, Leung B, Squire JA, Hill M, Arima N, Goss P, Hogg D, Mills GB. Identification, cloning, and characterization of a novel human T-cell-specific tyrosine kinase located at the hematopoietin complex on chromosome 5q. Blood. 1993;82:1561-1572.[Abstract/Free Full Text]

6. de Weers M, Verschuren MC, Kraakman ME, Mensink RG, Schuurman RK, van Dongen JJ, Hendriks RW. The Bruton's tyrosine kinase gene is expressed throughout B cell differentiation, from early precursor B cell stages preceding immunoglobulin gene rearrangement up to mature B cell stages. Eur J Immunol. 1993;23:3109-3114.[Medline] [Order article via Infotrieve]

7. Sommers CL, Huang K, Shores EW, Grinberg A, Charlick DA, Kozak CA, Love PE. Murine txk: a protein tyrosine kinase gene regulated by T cell activation. Oncogene. 1995;11:245-251.[Medline] [Order article via Infotrieve]

8. Tamagnone L, Lahtinen I, Mustonen T, Virtaneva K, Francis F, Muscatelli F, Alitalo R, Smith CI, Larsson C, Alitalo K. BMX, a novel nonreceptor tyrosine kinase gene of the BTK/ITK/TEC/TXK family located in chromosome Xp22.2. Oncogene. 1994;9:3683-3688.[Medline] [Order article via Infotrieve]

9. Kaipainen A, Korhonen J, Pajusola K, Aprelikova O, Persico MG, Terman BI, Alitalo K. The related FLT4, FLT1, and KDR receptor tyrosine kinases show distinct expression patterns in human fetal endothelial cells. J Exp Med. 1993;178:2077-2088.[Abstract/Free Full Text]

10. Kaukonen J, Lahtinen I, Laine S, Alitalo K, Palotie A. Bmx tyrosine kinase gene is expressed in granulocytes and myeloid leukemias. Br J Haematol. 1996;94:455-460.[Medline] [Order article via Infotrieve]

11. Tsukada S, Simon MI, Witte ON, Katz A. Binding of beta gamma subunits of heterotrimeric G proteins to the PH domain of Bruton tyrosine kinase. Proc Natl Acad Sci U S A. 1994;91:11256-11260.[Abstract/Free Full Text]

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