Expression of isoforms and splice variants of the latent transforming growth factor beta binding protein (LTBP) in cultured human liver myofibroblasts.
OBJECTIVE
The activation of hepatic stellate cells (HSC) to extracellular matrix (ECM) producing myofibroblasts (MFB) is the key pathogenetic event in human liver fibrogenesis. Latent transforming growth factor beta binding protein (LTBP), a component of the profibrogenic large latent transforming growth factor (TGF)-beta complex, is suggested to be important for secretion, latency, storage and activation of TGF-beta in the ECM. This study was performed to identify the expression profile of all hitherto known LTBP isoforms and LTBP splice variants in conjunction with that of TGF-beta isoforms in cultured human liver MFB.


METHODS
Cultured human MFB were analyzed for TGF-beta and LTBP using reverse-transcription polymerase chain reaction (RT-PCR), sequence analysis, immunofluorescence staining, metabolic labeling, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).


RESULTS
Transcripts of all three TGF-beta isoforms, of all four LTBP isoforms and of nearly all splice variants of LTBP-1 and LTBP-4 so far known were detected. Metabolic labeling followed by immunoprecipitation with anti-LTBP-1 antibody revealed the synthesis of LTBP proteins. Secretion of free LTBP and LTBP integrated into the large latent TGF-beta complex was demonstrated by size-exclusion chromatography. Co-localization of LTBP-1 and -2 with fibronectin and collagen type I was observed by double immunofluorescence staining.


CONCLUSIONS
The expression of a complete profile of hitherto known LTBP proteins by cultured human MFB suggests a role in modulating the bioactivity of TGF-beta in the diseased liver.
Mangasser-Stephan
K
K
Institute of Clinical Chemistry and Pathobiochemistry, Central Laboratory, RWTH-University Hospital, Aachen, Germany.
Gartung
C
C
Lahme
B
B
Gressner
A M
AM
D017398:Alternative Splicing; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D002478:Cells, Cultured; D002850:Chromatography, Gel; D003094:Collagen / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D053779:Latent TGF-beta Binding Proteins; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D011233:Precipitin Tests; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics; D012313:RNA / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D016212:Transforming Growth Factor beta / Q000096:biosynthesis / Q000235:genetics
D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D017931:DNA Primers; D005353:Fibronectins; D047908:Intracellular Signaling Peptides and Proteins; C509032:LTBP1 protein, human; C509040:LTBP4 protein, human; D053779:Latent TGF-beta Binding Proteins; D020033:Protein Isoforms; D016212:Transforming Growth Factor beta; D012313:RNA; D003094:Collagen