Differential expression of SM22 isoforms in myofibroblasts and smooth muscle cells from rabbit bladder.
The E-11 and 1-B8 monoclonal antibodies raised to the smooth muscle (SM)-specific SM22 protein from pig stomach were used to study the in vivo and in vitro phenotypic characteristics of myofibroblasts (MF) and SM cells (SMC) from the bladder detrusor muscle and serosal thickening of male rabbit. The 22-kDa SM22 band found in the SM extract appeared to be composed of distinct isoforms when examined in non-equilibrium two-dimensional gel electrophoresis (2D-EF): alpha (the most basic), beta, gamma, and delta (the most acidic) in the ratio of 34(alpha):23(beta):36(gamma):8(delta). Western blots of 2D-electrophoresed bladder extracts treated with E-11 and 1-B8 showed that alpha, beta, and delta, but not gamma isoforms were labeled with E-11, whereas alpha, beta, and gamma isoforms were stained with 1-B8. This differential immunoreactivity was not influenced by phosphorylation. The tissue distribution of SM22 immunostaining was heterogeneous in the bladder SM and serosal thickening developed as a consequence of partial outflow obstruction of the urinary bladder. At the cellular level, the 1-B8 and E-11 antibodies stained the SMC in a "diffuse" (the whole cytoplasm) and "honeycomb" (the peripheral cytoplasm) manner, whereas MF immunostaining was quite homogeneous. The two antibodies also reacted with cultured primary bladder SMC and MF grown in low serum conditions showing a heterogeneous SM22 cell distribution but an identical subcellular localization, i.e., the actin-containing filamentous network, distinguishable in part from that found in vivo. The immunocytochemical, Western blotting and 2D-EF patterns of MF from thickened serosa indicated that the gamma isoform alone is expressed in this tissue. This SM22 variant appeared before the completion of the cellular transition from MF to fully differentiated SMC. This pattern is reminiscent of bladder ontogenesis where SM22 expression in the developing bladder wall precedes that of SM myosin. Taken together these data suggest that: (i) SM22 isoforms are differently assorted in MF vs. SMC; (ii) SM22 is a SMC-lineage marker inasmuch as its expression occurs in an experimental condition characterized by a time-related cell phenotypic transition from MF to SMC, and (iii) cell conversion ability of serosal cells in the adult might take place via the reactivation of a specific "foetal" gene programme.
Department of Biomedical Sciences, University of Padua, Italy.
A
A
Chiavegato
M
M
Roelofs
R
R
Franch
E
E
Castellucci
F
F
Sarinella
S
S
Sartore
D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000737:chemistry; D019084:Fluorescent Antibody Technique, Indirect; D008297:Male; D008840:Microfilament Proteins; D009124:Muscle Proteins / Q000032:analysis*; D009130:Muscle, Smooth / Q000737:chemistry*; D011817:Rabbits; D001743:Urinary Bladder / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D008840:Microfilament Proteins; D009124:Muscle Proteins; C058992:Tagln protein, mouse; C051615:transgelin