Protein kinase C beta modulates thrombin-induced Ca2+ signaling and endothelial permeability increase.
We investigated the function of the Ca2+-dependent protein kinase C (PKC) beta1 in the regulation of endothelial barrier property. Human dermal microvascular endothelial cells (HMEC-1) were transduced with full-length PKCbeta1 antisense (AS) cDNA or control pLNCX vector to generate stable cell lines (HMEC-AS and HMEC-pLNCX, respectively). Analyses indicated that HMEC-AS expressed the antisense PKCbeta1 transcript with decreased PKCbeta protein level (without a change in PKCalpha or PKCepsilon). The baseline transendothelial 125I-albumin clearance rates of HMEC-1, HMEC-pLNCX, and HMEC-AS were 5.0+/-0.5 x 10(-2), 6.8+/-0.4 x 10(-2), and 6.9+/-0.6 x 10(-2) microl/min, respectively. Activation of HMEC-1 and HMEC-pLNCX with phorbol 12-myristate 13-acetate (PMA) increased the rates to the respective 14.5+/-1.7 x 10(-2) microl/min and 16.9+/-2.8 x 10(-2) microl/min (corresponding to 191% and 149% increases over baseline). However, in HMEC-AS, PMA increased the rate to 9.8+/-1.0 x 10(-2) microl/min (42%). When HMEC-1 and HMEC-pLNCX were activated with thrombin, the rates increased to 10.8+/-1.4 x 10(-2) and 14.0+/-1.9 x 10(-2) microl/min, respectively (116% and 106%). In contrast, thrombin stimulation of HMEC-AS more than doubled the increase to 27.2+/-3.5 x 10(-2) microl/min (294%). Furthermore, the thrombin-induced peak increase in the [Ca2+]i in HMEC-AS was greater than in control cells. Fluorescence-activated cell sorter analysis of thrombin receptor expression indicated that the augmented thrombin-induced responses were not attributable to altered receptor density in HMEC-AS. These results indicate that PKCbeta functions in a negative feedback manner to inactivate thrombin-generated signals and thereby modulates the endothelial permeability increase. Because decreased PKCbeta expression significantly reduced the PMA-induced permeability increase, PKCbeta may downregulate thrombin receptor function upstream of PKC activation (i.e., Ca2+).
Vuong
P T
PT
Department of Pharmacology, University of Illinois at Chicago, College of Medicine, 60607-7174, USA.
Malik
A B
AB
Nagpala
P G
PG
Lum
H
H
D000418:Albumins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002199:Capillary Permeability / Q000502:physiology*; D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D005246:Feedback; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000502:physiology*; D011493:Protein Kinase C / Q000235:genetics / Q000502:physiology*; D064546:Protein Kinase C beta; D016372:RNA, Antisense / Q000032:analysis; D018179:Receptors, Thrombin / Q000032:analysis; D015398:Signal Transduction / Q000502:physiology; D012867:Skin; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*
D000418:Albumins; D007527:Isoenzymes; D016372:RNA, Antisense; D018179:Receptors, Thrombin; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium