Tissue-specific expression and alternative splicing of fibroblast activation protein alpha (FAPα) gene in human stromal cells

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BACKGROUND: Activated stromal cells, responsible for tissue repair and restoration of tissue integrity, play an important role in tissue response to damage. The key marker of activated stromal cells is the fibroblast activation protein α (FAPα), which is apparently involved in the regulation of stromal cell functions, especially in the development of fibrosis. However, It is poorly known about tissue differences in expression and possible splice variants of this protein in stromal cells.

AIM: The aim is to establish the tissue specificity of FAPα expression, including the synthesis of alternative splicing products, in human stromal cells isolated from various sources.

METHODS: The expression of individual sites encoding the functional domains of FAPα in human stromal cells isolated from five tissue sources differing in embryonic origin and reparative reactions to damage (skin, subcutaneous adipose tissue, orbital adipose tissue, lungs, endometrium) was analyzed by real-time polymerase chain reaction. The key profibrotic factor TGFβ was used to activate stromal cells.

RESULTS: Low background expression of all RNA sequences encoding functional domains of FAPα responsible for protein configuration or for its enzymatic functions is noted in stromal cells from skin, lungs and adipose tissue. Their expression is significantly increased (2–2.5 times) upon induction of stromal cell activation by TGFβ. In endometrial mesenchymal stromal cell, the expression of these sites is extremely low and comparable to the level of expression in epithelial cells, which indicates the presence of tissue-specific expression of the FAPα gene in stromal cells. In all stromal cell lines, an increase in the relative expression level of the RNA sequence encoding the transmembrane domain of FAPα is observed.

CONCLUSION: Thus, the obtained results suggest that the analyzed cell lines either lack alternative FAPα splicing, or manifest themselves with a very low probability when activated in the form of a relative increase in the expression of the gene region encoding the transmembrane domain of FAPα. Most probably, the studied stromal cells synthesize mRNA carrying all the original exons and encoding predominantly the complete FAPα protein, that includes all functional domains.

Sobre autores

Anastasiya Tolstoluzhinskaya

Lomonosov Moscow State University

Autor responsável pela correspondência
Email: tolstoluzhinskayaae@my.msu.ru
ORCID ID: 0000-0001-8362-2902
Código SPIN: 6592-9889
Rússia, Moscow

Natalya Basalova

Lomonosov Moscow State University

Email: basalovana@my.msu.ru
ORCID ID: 0000-0002-2597-8879
Código SPIN: 2448-4671

Cand. Sci. (Biology)

Rússia, Moscow

Maxim Karagyaur

Lomonosov Moscow State University

Email: m.karagyaur@mail.ru
ORCID ID: 0000-0003-4289-3428
Código SPIN: 9504-4257

Cand. Sci. (Biology)

Rússia, Moscow

Anastasia Efimenko

Lomonosov Moscow State University

Email: efimenkoan@gmail.com
ORCID ID: 0000-0002-0696-1369
Código SPIN: 5110-5998

MD, Cand. Sci. (Medicine)

Rússia, Moscow

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2. Fig. 1. Structure of fibroblast activation protein alpha: a — protein sequence of the FAPα: amino acids 1–4 are responsible for the intracellular domain; 5–25 make up the transmembrane domain; the remaining amino acids up to 760 are the extracellular part of the protein, while the β-propeller domain consists of amino acids 54–492, and the α/β-hydrolase domain is 493–760; b — protein structure, side.

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3. Fig. 2. Micrographs of used cell cultures, phase contrast (inverted microscope "Leica DM IL Led", Leica Microsystems, Switzerland): a — dSC (stromal cells derived from human dermis); b — lSC (stromal cells derived from human lungs); c — aSC (stromal cells derived from human adipose tissue); d — eSC (stromal cells derived from menstrual secretions of the endometrium of the uterus); e — oaSC (stromal cells derived from orbital adipose tissue); f — HEK (HEK-293T) are linear cells of human embryonic renal epithelium. The scale bar is 100 microns.

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4. Fig. 3. Electrophoresis of complementary DNA polymerase chain reaction products of the lSC line (lung stromal cells). The primers the reactions were associated with certain regions of the FAPα gene encoding various functional domains of the protein (from left to right): 1 — GAPDH is a constitutive gene for normalization of results (glyceraldehyde-3-phosphate dehydrogenase), 2 — intracellular domain, 3 — transmembrane domain, 4 — extracellular domain, 5 — β-propeller domain, 6 — α/β-hydrolase domain, and further GeneRuler 100 bp DNA Ladder indicating 300, 200 and 100 base pairs (п.о.).

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5. Fig. 4. Graphs show the expression level of FAPα gene fragments (in conventional units) in stromal cell cultures isolated from various tissue sources: lung (лСК), skin (дСК), orbital fat (оСК), abdominal fat (жСК), endometrium (эСК), and the HEK line. The studied FAPα fragments encode various functional domains of the protein: a — intracellular, b — transmembrane, c — extracellular, d — β-propeller and e — α/β-hydrolase.

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6. Fig. 5. Graphs show the expression level of FAPα gene fragments (in conventional units) in cell cultures before (blue columns) and after (red columns) addition of the TGFß factor: a — lSC (pulmonary stromal cells), b — dSC (dermal stromal cells), c — aSC (stromal cells derived from adipose tissue). The studied gene sequences encode different functional domains, respectively: 1 — intracellular domain, 2 — transmembrane domain, 3 — extracellular domain, 4 — β-propeller domain, 5 — α/β-hydrolase domain. These numbers are the value of p, which was obtained as a result of calculating the Mann–Whitney criterion for a pair of samples without and with the addition of TGFβ and the Kraskel–Wallis criterion for groups of samples without the addition of TGFβ and after the addition of TGFβ.

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7. Fig. 6. Graphs show the expression level of FAPα gene fragments (in conventional units) in cell cultures: a — stromal cells derived from orbital fat (oaSC); b — stromal cells derived from the endometrium of the uterus (eSC). The studied gene sequences encode different functional domains, respectively: 1 — intracellular domain, 2 — transmembrane domain, 3 — extracellular domain, 4 — β-propeller domain, 5 — α/β-hydrolase domain. The p value is calculated according to the Kraskel–Wallis criterion.

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8. Fig. 7. Electrophoresis of PCR products obtained from complementary DNA samples of dermal stromal cells (дСК) and pulmonary stromal cells (лСК) without and after addition of TGFβ, as well as endometrial stromal cells (эСК). The rightmost band is DNA Ladder 1 kb DNA length markers (NL001; “Eurogene”, Russia). From left to right: the first five bands after the marker are the products of amplification of the whole FAPα gene; the next three bands are PCR products of samples of pulmonary stromal cells before and after induction of TGFβ, obtained using pairs of primers No. 1–3 of Table 2, respectively; the next three bands are PCR products of endometrial stem cell samples obtained using primer pairs No. 1, No. 2 and No. 3. The yellow numbers indicate the bands selected for sequencing. PCR — polymerase chain reaction, “–TGFß” — without addition of TGFß, “+TGFß” — after addition of TGFß.

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