Coexpression of CD9 augments the ability of membrane-bound heparin-binding epidermal growth factor-like growth factor (proHB-EGF) to preserve renal epithelial cell viability.
BACKGROUND
Transfection of renal epithelial cells (NRK 52E) with membrane-associated heparin-binding epidermal growth factor-like growth factor (proHB-EGF) increased renal epithelial cell survival by promoting cell-cell and cell-extracellular matrix interactions. ProHB-EGF has been shown to form a complex in the plasma membrane with the tetraspanin CD9, an interaction that significantly increases the effectiveness of proHB-EGF as a juxtacrine mitogenic agent.


METHODS
We examined whether the coexpression of proHB-EGF and CD9 would increase renal epithelial cell survival. CD9 was stably transfected into NRK 52E cells, either alone (NRKCD9) or together with proHB-EGF (NRKboth).


RESULTS
Juxtacrine mitogenic activity of NRKCD9 was no different than in cells transfected with vector alone (NRKvector), but was increased by NRKboth; juxtacrine mitogenic activity by NRKboth was twofold greater than when proHB-EGF was transfected alone (NRKproHB-EGF). When grown in 10% fetal calf serum, growth rates were similar among all transfectants. However, in 1% fetal calf serum, NRKproHB-EGF grew 50% faster than NRKvector or NRKCD9, and NRKboth grew 20% to 50% faster than NRKproHB-EGF at one, two, and three days of culture. NRKproHB-EGF attachment to plastic substratum at one, two, and three hours was 250% greater than that of NRKvector, and NRKboth was 20% to 30% greater than that of NRKproHB-EGF. Coating plates with either poly 2-hydroxyethyl methacrylate or the GRGDTP peptide prevented normal cell-extracellular matrix attachment, and NRKvector or NRKCD9 failed to attach or form cell-cell attachments. NRKproHB-EGF exhibited 300% and NRKboth exhibited 600% greater cell viability under these conditions. Expression of type I and type III collagen mRNA was enhanced similarly in NRKproHB-EGF and NRKboth, but the expression of beta1 integrin was up-regulated only in NRKboth.


CONCLUSIONS
Coexpression of proHB-EGF and CD9 may render the renal epithelial cells more resistant to disruption of cell-cell and cell-matrix interactions and could accelerate the re-establishment of these attachments.
Takemura
T
T
Department of Pediatrics, Kinki University, Osaka, Japan, and Department of Medicine, Vanderbilt University, Nashville, Tennessee, USA.
Hino
S
S
Murata
Y
Y
Yanagida
H
H
Okada
M
M
Yoshioka
K
K
Harris
R C
RC
D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D017209:Apoptosis; D001483:Base Sequence; D002448:Cell Adhesion; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D017931:DNA Primers / Q000235:genetics; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D015870:Gene Expression; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D007668:Kidney / Q000166:cytology* / Q000378:metabolism*; D008562:Membrane Glycoproteins; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D060245:Tetraspanin 29; D014162:Transfection
D015703:Antigens, CD; D017931:DNA Primers; C587885:Hbegf protein, rat; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D011498:Protein Precursors; D012333:RNA, Messenger; D060245:Tetraspanin 29; C042652:epidermal growth factor precursor; D004815:Epidermal Growth Factor