Phorbol 12,13-dibutyrate Treatment Research Articles

Distinct protein kinase C isoforms drive the cell cycle re-entry of two separate populations of neonatal rat ventricular cardiomyocytes.

The present study tested the hypothesis that protein kinase C-α (PKC-α) recruitment in the presence of the p38α/β MAPK inhibitor SB203580 facilitated the appearance and cell cycle re-entry of nestin(+)-neonatal rat ventricular cardiomyocytes (NNVMs) and induced a transcript profile delineating a proliferative phenotype. Phorbol 12,13-dibutyrate (PDBu) treatment did not induce de novo nestin expression or increase the cell cycle re-entry of 1-day-old NNVMs but significantly increased runt-related transcription factor 1 (Runx1) and p16 cell cycle inhibitor (CDKN2a) mRNA levels and downregulated epithelial cell transforming 2 (ECT2) mRNA expression. SB203580 administration to PDBu-treated NNVMs induced de novo nestin expression, preferentially increased the density (normalized to 500 NNVMs) of nestin(+)-NNVMs that incorporated 5-bromo-2'-deoxyuridine (PDBu, 1.4 ± 3 vs. PDBu/SB203580, 128 ± 34; n = 5 independent litters), significantly inhibited CDKN2a and Runx1 mRNA upregulation and reversed ECT2 mRNA downregulation. PDBu treatment of NNVMs reduced PKC-α, protein kinase-δ (PKC-δ) and protein kinase-ε (PKC-ε) protein levels and GF109203X (conventional PKC isoform inhibitor) selectively attenuated PKC-α protein downregulation. GF109203X administration to PDBu/SB203580-treated NNVMs significantly reduced the density of nestin(+)-NNVMs that incorporated 5-bromo-2'-deoxyuridine (PDBu/SB203580/GF109203X, 40 ± 46; n = 5). Moreover, GF109203X/PDBu/SB203580 treatment unmasked the predominant appearance of a separate NNVM population that incorporated 5-bromo-2'-deoxyuridine (PDBu/SB203580/GF109203X, 192 ± 42; n = 5) delineated by the absence of de novo nestin expression. Sotrastaurin (conventional/novel PKC isoform inhibitor) administration to PDBu/SB203580-treated NNVMs significantly attenuated the density of nestin(+)-NNVMs (PDBu/SB203580/sotrastaurin, 8 ± 10; n = 4) and nestin(-)-NNVMs (PDBu/SB203580/sotrastaurin, 64 ± 30; n = 4) that incorporated 5-bromo-2'-deoxyuridine. These data reveal that the neonatal rat heart contains at least two separate populations of NNVMs that re-enter the cell cycle and the preferential appearance of nestin(+)- or nestin(-)-NNVMs is driven by distinct PKC isoforms in the presence of SB203580.NEW & NOTEWORTHY The appearance of nestin(+)-neonatal rat ventricular cardiomyocytes that re-entered the cell cycle following phorbol ester stimulation in the presence of p38α/β MAPK inhibitor SB203580 was associated with the inhibition of Runx1 and CDKN2a mRNA upregulation. PKC-α selectively induced the cell cycle re-entry of nestin(+)-neonatal rat ventricular cardiomyocytes. Pharmacological inhibition of PKC-α with concomitant p38α/β MAPK suppression unmasked the cell cycle re-entry of a second population of neonatal rat ventricular cardiomyocytes in the absence of nestin expression.

Nestin Expressed by Pre-Existing Cardiomyocytes Recapitulated in Part an Embryonic Phenotype; Suppressive Role of p38 MAPK.

Nestin(+) -cardiomyocytes were identified in the ischemically damaged human/rodent heart, albeit the cellular source, and signaling events implicated in the appearance of the intermediate filament protein remained undefined. Expression of the enhanced green fluorescent protein (EGFP) driven by the second intron of the nestin gene identified a subpopulation of EGFP/nestin(+) cells that differentiated to a vascular phenotype in the peri-infarct/infarct region of post-MI mice albeit the transgene was not detected in nestin(+) -cardiomyocytes. α-MHC-driven expression of the reporter mCherry was detected in troponin-T(+) - and nestin(+) -cardiomyocytes in the peri-infarct/infarct region of post-MI mice. However, the cell cycle re-entry of nestin/mCherry(+) -cardiomyocytes was not observed. Nestin staining was identified in a paucity of neonatal rat ventricular cardiomyocytes (NNVM). Exposure to phorbol 12,13-dibutyrate (PDBu) induced NNVM hypertrophy but did not promote nestin expression or Brdu incorporation. PDBu treatment of NNVMs phosphorylated p38 MAPK and HSP27 and HSP27 phosphorylation was abrogated by the p38 MAPK inhibitor SB203580. PDBu/SB203580 co-treatment significantly increased the percentage of NNVMs that expressed nestin and incorporated Brdu. In the heart of embryonic 10.5 day mice, nestin immunoreactivity was observed in cycling troponin-T(+) -cardiomyocytes. Nestin was also detected in embryonic rat ventricular cardiomyocytes and depletion of the intermediate filament protein attenuated cell cycle re-entry. Thus, nestin expressed by pre-existing cardiomyocytes following ischemic damage recapitulated in part an embryonic trait and may provide the requisite phenotype to initiate cell cycle re-entry. However, the overt activation of the p38 MAPK pathway post-MI may in part limit the appearance and inhibit the cell cycle re-entry of nestin(+) -cardiomyocytes. J. Cell. Physiol. 232: 1717-1727, 2017. © 2016 Wiley Periodicals, Inc.

Phorbol Dibutyrate (PDB) Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) May Involve Down‐Regulation of MicroRNAs (miRs)

In colonic epithelia, the concerted action of apical membrane Cl− channels and basolateral membrane Na+/K+‐ATPase, Na+‐K+‐2Cl− cotransporters and K+ channels, account for net Cl− secretion. CFTR, the major apical Cl− channel, is regulated by protein kinases (PK) A and C. We had previously showed in human colonic T84 cells that the cAMP activator, prostaglandin E1 (PGE1,10nM), stimulated Cl− secretion via CFTR, measured as short circuit current (ΔISc, μA/cm2) and this was attenuated by both acute and prolonged pretreatment with the PKC activator, phorbol dibutyrate (PDB, 100nM). Further, 2h of pretreatment with PDB significantly reduced CFTR mRNA. Since microRNAs (miRs) are known to modulate proteins by either degrading mRNA, or by inhibiting translation, we initially tested whether 6 specific CFTR related miRs: −145, −331, −384, −494, −1246 and 1290 were regulated by PDB. However, the expressions of these miRs were not significantly altered by PDB (FASEB 29:855.5,’15). In this study, we further explored the molecular basis of the attenuation in Cl− secretion. We first confirmed, by Western blotting, that CFTR protein expression is down regulated at 2h PDB treatment (n=2). In addition, the mRNA (60%, n=3) and protein expression for NKCC was also decreased. Since CFTR 3′ UTR contains almost 500 recognized miR target sites, we next undertook a detailed algorithm screening process involving TargetScan, miRDB, EMBL‐EBI and Diana to identify further possible CFTR miR regulators. We hypothesized that the miRs identified through algorithmic screening would be up‐regulated after PDB treatment, leading to a decrease in CFTR expression. We specifically screened miRs: −362, −433, −509, −600, −607, −619, −631, and −944, which were identified by at least 3 algorithms. After treating T84 cells with ±100nM PDB for 2h, 6h, and 24h, RNA was isolated using TriZol Reagent and cDNA was synthesized using an Invitrogen cDNA synthesis kit. We compared the expression levels of these miRs using an Invitrogen miRNA qRT‐PCR Kit with U6 snRNA as an internal control. Based on three separate experiments each (p<0.05), the results show that miR −607 (Control: 1.37±0.18, 2h: 0.51±0.08, 6h: 0.77±0.09, 24h: 0.55±0.18), miR‐619 (Control: 2.04±0.92, 2h: 0.58±0.05, 6h: 0.42±0.06, 24h: 0.44±0.04), and miR‐944 (Control: 1.50±0.30, 2h: 0.46±0.07, 6h: 0.36±0.08, 24h: 0.40±0.13) expressions were significantly down‐regulated at all measured time‐points. miR‐631 (Control: 1.24±0.18) expression was significantly down‐regulated at 2h (0.60±0.05) and 6h (0.58±0.04) but not at 24h (0.78±0.22). The results with miR‐607 are in contrast to studies in Caco2 cells, where an increase in miR‐607 leads to a decrease in CFTR expression (Biochem J. 438, p.25–32 2011). The other miRs tested did not show significant alteration of expression at any measured time‐point. Thus, PDB treatment reduces CFTR and NKCC expression and down regulates specific miRs. We speculate that perhaps there is an intermediary protein, the target of miRs: −607, −619, and −944, that alters CFTR/NKCC expression.Support or Funding InformationAmerican Physiological Society Undergraduate Research Excellence Fellowship (UGREF)

Protein Kinase Cδ-mediated Phosphorylation of Connexin43 Gap Junction Channels Causes Movement within Gap Junctions followed by Vesicle Internalization and Protein Degradation

Phosphorylation of gap junction proteins, connexins, plays a role in global signaling events involving kinases. Connexin43 (Cx43), a ubiquitous and important connexin, has several phosphorylation sites for specific kinases. We appended an imaging reporter tag for the activity of the δ isoform of protein kinase C (PKCδ) to the carboxyl terminus of Cx43. The FRET signal of this reporter is inversely related to the phosphorylation of serine 368 of Cx43. By activating PKC with the phorbol ester phorbol 12,13-dibutyrate (PDBu) or a natural stimulant, UTP, time lapse live cell imaging movies indicated phosphorylated Ser-368 Cx43 separated into discrete domains within gap junctions and was internalized in small vesicles, after which it was degraded by lysosomes and proteasomes. Mutation of Ser-368 to an Ala eliminated the response to PDBu and changes in phosphorylation of the reporter. A phosphatase inhibitor, calyculin A, does not change this pattern, indicating PKC phosphorylation causes degradation of Cx43 without dephosphorylation, which is in accordance with current hypotheses that cells control their intercellular communication by a fast and constant turnover of connexins, using phosphorylation as part of this mechanism.

Phosphorylation of G Protein–Coupled Receptor Kinase 2–Interacting Protein 1 Tyrosine 392 Is Required for Phospholipase C-γ Activation and Podosome Formation in Vascular Smooth Muscle Cells

Podosomes, which are actin-rich structures, contribute to cell motility, matrix remodeling, and tissue remodeling. We have shown that G protein-coupled receptor kinase 2-interacting protein 1 (GIT1) colocalizes with podosomes and is important in podosome formation in endothelial cells. Src stimulates GIT1 tyrosine phosphorylation, which is critical for phospholipase C-γ (PLCγ) activation. In this study, we identified specific GIT1 tyrosines required for PLCγ activation and podosome formation in vascular smooth muscle cells (VSMC). We used phorbol 12,13-dibutyrate (PDBU) to induce podosomes in A7r5 VSMC. GIT1 colocalized with podosomes and GIT1 knockdown using short interfering RNA significantly reduced podosome formation. PDBU stimulated GIT1 tyrosine phosphorylation. GIT1 tyrosine phosphorylation was dramatically decreased in SYF-/- cells, and it was also reduced by pretreatment with the protein kinase C (PKC) and Src inhibitors, suggesting that GIT1 phosphorylation was dependent on PKC and Src. By mutation analysis of multiple tyrosines, we found that PDBU specifically increased GIT1-Y392 phosphorylation. Overexpression of GIT1 (Y392F) but not of GIT1 (Y321F) decreased PDBU-mediated PLCγ activation and podosome formation without effect on extracellular signal-regulated kinase 1/2 activation. Additionally, we provide evidence that GIT1 knockout VSMC have markedly fewer podosomes on PDBU treatment compared with wild-type VSMC. These data show that GIT1 is a key regulator of podosome formation in VSMC. In conclusion, our data suggest that GIT1-Y392 phosphorylation is critical for PDBU-induced podosome formation by regulating PLCγ activation. We propose that specific signaling modules are assembled in a GIT1 phosphotyrosine-dependent manner as exemplified by PLCγ activation versus extracellular signal-regulated kinase 1/2 activation.

Rapid Activation of Dormant Presynaptic Terminals by Phorbol Esters

Presynaptic stimulation stochastically recruits transmission according to the release probability (P(r)) of synapses. The majority of central synapses have relatively low P(r), which includes synapses that are completely quiescent presynaptically. The presence of presynaptically dormant versus active terminals presumably increases synaptic malleability when conditions demand synaptic strengthening or weakening, perhaps by triggering second messenger signals. However, whether modulator-mediated potentiation involves recruitment of transmission from dormant terminals remains unclear. Here, by combining electrophysiological and fluorescence imaging approaches, we uncovered rapid presynaptic awakening by select synaptic modulators. A phorbol ester phorbol 12,13-dibutyrate (PDBu) (a diacylglycerol analog), but not forskolin (an adenylyl cyclase activator) or elevated extracellular calcium, recruited neurotransmission from presynaptically dormant synapses. This effect was not dependent on protein kinase C activation. After PDBu-induced awakening, these previously dormant terminals had a synaptic P(r) spectrum similar to basally active synapses naive to PDBu treatment. Dormant terminals did not seem to have properties of nascent or immature synapses, judged by NR2B NMDAR (NMDA receptor) receptor subunit contribution after PDBu-stimulated awakening. Strikingly, synapses rendered inactive by prolonged depolarization, unlike basally dormant synapses, were not awakened by PDBu. These results suggest that the initial release competence of synapses can dictate the acute response to second messenger modulation, and the results suggest multiple pathways to presynaptic dormancy and awakening.

Phorbol 12,13-dibutyrate-mediated contraction of the stimulated bronchial smooth muscles of mice

Increasing evidence suggests that protein kinase C (PKC) is involved in the Ca(2+) sensitization of various smooth muscle contractions. However, the exact role of PKC on bronchial smooth muscle (BSM) contraction is still unclear. In the present study, to determine the role of PKC activation in the BSM contraction, the effects of phorbol 12,13-dibutyrate (PDBu) on BSM tone were examined in the absence and presence of contractile stimulation. Although PDBu had no effect on the basal tone, the contraction induced by acetylcholine, high K(+) depolarization or Ca(2+) ionophore A23187 was significantly augmented by PDBu. Western blot analyses also revealed that the increase in the level of phosphorylated myosin light chain (MLC) induced by high K(+) depolarization was significantly augmented by PDBu treatment. Interestingly, neither high K(+) depolarization alone nor PDBu alone caused CPI-17 phosphorylation, but a significant phosphorylation of CPI-17 was observed when BSMs were co-stimulated by high K(+) and PDBu. Thus, a certain level of intracellular Ca(2+) might be needed both for an activation of CPI-17 and an induction of contraction induced by PDBu in mouse BSMs.

Up-Regulation of Cell Surface Insulin Receptor by Protein Kinase C-α in Adrenal Chromaffin Cells

Our previous study showed that treatment of cultured bovine adrenal chromaffin cells with phorbol 12, 13-dibutyrate (PDBu) or 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a rapid ( 15 h) translocation of both conventional (c) protein kinase C-α (PKC-α) and novel PKC-e (but not atypical PKC-ζ) from cytosol to membranes, whereas thymeleatoxin (TMX) increased the similar but selective membrane association of only cPKC-α. In the present study, chronic (≥12 h) treatment of chromaffin cells with PDBu raised cell surface 125I-insulin binding without altering the KD value ; it developed in a concentration (EC50 = 1.9 nM)-and time (t1/2 = 14.6 h)-dependent manner, reaching its maximum 115% increase at 48 h. Either TPA (30 nM) or TMX (EC50 = 6.4 nM) also increased 125I-insulin binding by 97 or 88%, whereas the biologically inactive 4α-TPA had no effect. The increasing effect of PDBu (30 nM for 24 h) on 125I-insulin binding was significantly blocked, even when H7, an inhibitor of PKC, was added at 8 h after the initiation of PDBu treatment. Concurrent treatment with brefeldin A, an inhibitor of vesicular transport from the trans-Golgi network, cycloheximide, an inhibitor of protein synthesis, or 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, abolished the PDBu-induced increment of 125I-insulin binding. Western blot analysis, using antibody against the β-subunit of the insulin receptor, showed that treatment with PDBu (30 nM) or TMX (EC50 = 2.3 nM) increased levels of insulin receptor precursor (~190 kDa ; t1/2 = 7.1 h) and insulin receptor β-subunit (t1/2 = 15.4 h), causing their almost maximum 52 and 59% rises, respectively, at 24 h. Northern blot analysis revealed that PDBu or TMX increased levels of insulin receptor mRNAs by ~35% as soon as 3 h, producing its monophasic peak ~76% increases at 24 h. All of these increasing effects of PDBu and TMX on 125I-insulin binding and insulin receptor β-subunit and insulin receptor mRNA levels were entirely prevented by simultaneous treatment with Go6976, a selective inhibitor of cPKC. These results suggest that long-term activation of cPKC-α up-regulates the density of the cell surface insulin receptor via transcriptional/translational events.

Direct Protein Kinase C-dependent Phosphorylation Regulates the Cell Surface Stability and Activity of the Potassium Chloride Cotransporter KCC2

The potassium chloride cotransporter KCC2 plays a major role in the maintenance of transmembrane chloride potential in mature neurons; thus KCC2 activity is critical for hyperpolarizing membrane currents generated upon the activation of gamma-aminobutyric acid type A and glycine (Gly) receptors that underlie fast synaptic inhibition in the adult central nervous system. However, to date an understanding of the cellular mechanism that neurons use to modulate the functional expression of KCC2 remains rudimentary. Using Escherichia coli expression coupled with in vitro kinase assays, we first established that protein kinase C (PKC) can directly phosphorylate serine 940 (Ser(940)) within the C-terminal cytoplasmic domain of KCC2. We further demonstrated that Ser(940) is the major site for PKC-dependent phosphorylation for full-length KCC2 molecules when expressed in HEK-293 cells. Phosphorylation of Ser(940) increased the cell surface stability of KCC2 in this system by decreasing its rate of internalization from the plasma membrane. Coincident phosphorylation of Ser(940) increased the rate of ion transport by KCC2. It was further evident that phosphorylation of endogenous KCC2 in cultured hippocampal neurons is regulated by PKC-dependent activity. Moreover, in keeping with our recombinant studies, enhancing PKC-dependent phosphorylation increased the targeting of KCC2 to the neuronal cell surface. Our studies thus suggest that PKC-dependent phosphorylation of KCC2 may play a central role in modulating both the functional expression of this critical transporter in the brain and the strength of synaptic inhibition.

Differential actin isoform reorganization in the contracting A7r5 cell

In the present study, we investigated the reorganization of alpha- and beta-actin in the contracting A7r5 smooth muscle cell. The remodeling of these actin variants was markedly different in response to increasing concentrations of phorbol 12, 13-dibutyrate (PDBu). At the lowest concentrations (< or =10(-7) mol/L), cells showed an approximately 70% loss in alpha-actin stress fibers with robust transport of this isoform to podosomes. By comparison, beta-actin remained in stress fibers in cells stimulated at low concentrations (< or =10(-7) mol/L) of PDBu. However, at high concentrations (> or =10(-6)mol/L) approximately 50% of cells showed transport of beta-actin to podosomes. Consistent with these findings, staining with phalloidin indicated a significant decrease in the whole-cell content of F-actin with PDBu treatment. However, staining with DNase I indicated no change in the cellular content of G-actin, suggesting reduced access of phalloidin to tightly packed actin in the podosome core. Inhibition of protein kinase C (staurosporine, bisindolymaleimide) blocked PDBu-induced (5 x 10(-8) mol/L) loss in alpha-actin stress fibers or reversed podosome formation with re-establishment of alpha-actin stress fibers. By comparison, these inhibitors caused partial loss of beta-actin stress fibers. The results support our earlier conclusion of independent remodeling of alpha- and beta-actin cytoskeletal structure and suggest that the regulation of these structures is different.

Effect of PDBu on [alpha]1‐adrenoceptor‐mediated contraction and changes of intracellular free Ca2+ concentration in uterine artery of pregnant sheep

PKC activation by phorbol 12, 13-dibutyrate (PDBu) shows inhibitory effects on phenylephrine (PE)-induced contractions in pregnant uterine arteries. We hypothesize that PDBu inhibits PE-induced increases of intracellular free Ca2+ concentration ([Ca2+]i). Uterine arteries were isolated from near term pregnant ewes. Vascular smooth muscle [Ca2+]i was measured simultaneously with muscle contraction in the same tissue loaded with Fura-2 by using a CAF-110 intracellular Ca2+ analyzer. PE-induced contraction and simultaneously measured [Ca2+]i were compared between before and after PDBu treatment. Compared with before PDBu treatment, both 0.1 and 1 μM PDBu produced a significant decrease in PE-induced contraction and completely blocked PE-induced increases of [Ca2+]i as indicated by no changes of Rf340/380. These results suggested that PKC activation in pregnant uterine artery plays an inhibitory role in α1-adrenoceptor-mediated contraction through blocking the availability of free Ca2+ to the contractile proteins. (Supported in part by NIH grants HL57787 and HD 31226)

Differential actin isoform reorganization in the contracting A7r5 cell

In the present study, we investigated the reorganization of α- and β-actin in the contracting A7r5 smooth muscle cell. The remodeling of these actin variants was markedly different in response to increasing concentrations of phorbol 12, 13-dibutyrate (PDBu). At the lowest concentrations (≤10−7 mol/L), cells showed an ~70% loss in α-actin stress fibers with robust transport of this isoform to podosomes. By comparison, β-actin remained in stress fibers in cells stimulated at low concentrations (≤10−7 mol/L) of PDBu. However, at high concentrations (≥10−6 mol/L) ~50% of cells showed transport of β-actin to podosomes. Consistent with these findings, staining with phalloidin indicated a significant decrease in the whole-cell content of F-actin with PDBu treatment. However, staining with DNase I indicated no change in the cellular content of G-actin, suggesting reduced access of phalloidin to tightly packed actin in the podosome core. Inhibition of protein kinase C (staurosporine, bisindolymaleimide) blocked PD...

Regulation of a1‐adrenoceptor‐mediated contractions of the uterine artery by PKC in pregnant sheep

Little is known about the adaptation of contractile mechanisms of uterine arterial smooth muscle to pregnancy. We hypothesize that PKC plays an important role in the regulation of α1-adrenoceptor-mediated contractions of the uterine artery and their adaptation to pregnancy. Uterine arteries were isolated from pregnant ewes (~140 days gestation). α1-adrenoceptor-mediated contractions were measured with increasing concentrations of phenylephrine (PE) in half-log increments in the absence or presence of the PKC activator phorbol 12, 13-dibutyrate (PDBu) and/or PKC inhibitor GF-109203X (GF). PE produced dose-dependent contractions of uterine arteries. PDBu (0.3 and 1 μM) significantly decreased pD2 value and maximum contraction (Emax) induced by PE. GF itself (0.1, 0.3, 1 μM) markedly decreased pD2 value and Emax induced by PE in a dose dependent pattern. However, treatment with PDBu and 1 μM GF significantly increased pD2 and Emax as compared with PDBu treatment alone without GF. These results suggest that activation of PKC plays an inhibitory role in the regulation of α1-adrenoceptor-mediated contraction of the uterine arteries during late pregnancy, which is likely to be important in the adaptation of α1-adrenoceptor-mediated contractions to pregnancy. (Supported in part by NIH grants HL57787 and HD 31226)

Parathyroid hormone stimulates phosphatidylethanolamine hydrolysis by phospholipase D in osteoblastic cells

Parathyroid hormone (PTH) and phorbol-12,13-dibutyrate (PDBu) stimulate phospholipase D (PLD) activity and PC hydrolysis in UMR-106 osteoblastic cells {Singh, A.T., Kunnel, J.G., Strieleman, P.J., and Stern, P.H. (1999) Parathyroid Hormone (PTH)-(1-34), [Nle8,18,Tyr34]PTH-(3-34) Amide, PTH-(1-31) Amide, and PTH-Related Peptide-(1-34) Stimulate Phosphatidylcholine Hydrolysis in UMR-106 Osteoblastic Cells: Comparison with Effects of Phorbol 12,13-Dibutyrate, Endocrinology 140, 131-137}. The current studies were designed to determine whether ethanolamine-containing phospholipids, and specifically PE, could also be substrates. In cells labeled with 14C-ethanolamine, PTH and PDBu treatment decreased 14C-PE. In cells co-labeled with 3H-choline and 14C-ethanolamine, PTH and PDBu treatment increased both 3H-choline and 14C-ethanolamine release from the cells. Choline and ethanolamine phospholipid hydrolysis was increased within 5 min, and responses were sustained for at least 60 min. Maximal effects were obtained with 10 nM PTH and 50 nM PDBu. Dominant negative PLD1 and PLD2 constructs inhibited the effects of PTH on the phospholipid hydrolysis. The results suggest that both PC and PE are substrates for phospholipase D in UMR-106 osteoblastic cells and could therefore be sources of phospholipid hydrolysis products for downstream signaling in osteoblasts.

ER stress-induced caspase-12 activation is inhibited by PKC in neuronal cells

Caspase-12 is activated when the cells are exposed to excess levels of various stimuli, which cause endoplasmic reticulum (ER) stress. Protein kinase C (PKC) plays an important role in many signaling pathways in cells, and the activation of PKC has multiple actions in the signaling function of the ER. This study examined whether or not phorbol 12, 13-dibutyrate (PDBu)-induced PKC activation modulates caspase-12 cleavage and it's processing, using a wild type caspase-12 overexpressing neuronal cell line, known as Cas-12 cells. The thapsigargin treatment induced caspase-12 fragmentation in the Cas-12 cells. This was inhibited by PKC, which had previously been stimulated by PDBu. The PDBu treatment attenuated the ER stress-induced translocation of caspase-12 from the ER to the cytoplasm. The caspase-3 specific inhibitor blocked caspase-12 fragmentation, and purified caspase-12 was cleaved by the active caspase-3 in vitro, suggesting that caspase-12 might be a substrate for caspase-3. In addition, the PDBu treatment influenced the decrease of active caspase-3 fragment. These results suggest that an ER stress induces the activation of caspase-12 via caspase-3, and that PKC regulates both caspase-12 and caspase-3 activations in Cas-12 cells.

Podosome-mediated matrix resorption and cell motility in vascular smooth muscle cells

The migration of vascular smooth muscle cells (VSMCs) is a principal factor for the development and progression of vascular diseases. In addition, phenotypic alteration from the contractile (differentiated) to the synthetic (dedifferentiated) state and a proteolytic process in the form of extra cellular matrix degradation are necessary for SMC invasion. The actual mechanism leading to the focal degradation of basement membrane matrix components and, hence, SMC migration within the tissue itself is, however, unclear. In response to phorbol ester [phorbol-12,13-dibutyrate (PDBu)], VSMCs in culture form podosomes, dynamic organelles critical for cell adhesion and substrate degradation that are typically found in invasive cells and cells that cross tissue boundaries. Here, we show that PDBu-stimulated VSMCs resorb the extracellular matrix at the sites of podosomes. Podosome formation correlates with an increased polarization of VSMCs on fibronectin- or collagen-coated flexible substrates in addition to a concomitant induction of cell motility. VSMCs embedded in reconstituted basement membrane support adopt the typical spindle-shaped morphology of differentiated SMCs in vivo and, after PDBu treatment, form peripheral lamellipodia and podosomes around their matrix-contacting surface. Our findings demonstrate that podosome formation is the potential mechanism underlying the ability of VSMCs to traverse the surrounding basement membrane and escape the barrier of the tunica media in vascular diseases.

Dual Phospholipase C/Diacylglycerol Requirement for Protein Kinase D1 Activation in Lymphocytes

The serine/threonine kinase protein kinase D1 (PKD1) is a protein kinase C (PKC) substrate that mediates antigen receptor signal transduction in lymphocytes. PKC phosphorylates serines 744/748 within the PKD1 catalytic domain, and this is proposed to be necessary and sufficient for enzyme activation. Hence, a PKD1 mutant with alanine substituted at positions 744 and 748 (PKD-S744A/S748A) is catalytically inactive. Conversely, a PKD1 mutant with glutamic residues substituted at positions 744 and 748 as phospho-mimics (PKD-S744E/S748E) is constitutively active when expressed in Cos7 or HeLa cells. The present study reveals that Ser-744/Ser-748 phosphorylation is required for PKD1 activation in lymphocytes. However, PKD-S744E/S748E is not constitutively active but, like the wild type enzyme, requires antigen receptor triggering or phorbol ester stimulation. Antigen receptor activation of wild type PKD is dependent on phospholipase C (PLC)/diacylglycerol (DAG) and PKC, whereas PKD-S744E/S748E is only dependent on PLC/DAG but no longer requires PKC. Hence, substitution of serines 744 and 748 with glutamic residues as phospho-mimics bypasses the PKC requirement for PKD1 activation but does not bypass the need for antigen receptors, PLC, or DAG. In lymphocytes, PKD1 is, thus, not regulated by PLC and PKC in a linear pathway; rather, PKD1 activation has more stringent requirements for integration of dual PLC signals, one mediated by PKCs and one that is PKC-independent.

Protein kinase C regulates functional coupling of beta1-adrenergic receptors to Gi/o-mediated responses in cardiac myocytes.

The effect of protein kinase C (PKC) activation on beta1-adrenergic receptor (beta1-AR) regulation of the cardiac L-type Ca2+ current (ICa,L) was studied using the whole-cell patch clamp technique. Treatment of guinea pig ventricular myocytes with phorbol-12,13-dibutyrate (PDBu) caused a significant decrease in ICa,L sensitivity to stimulation by submaximal beta1-AR activation using isoproterenol (Iso). This decrease in sensitivity was also associated with the ability of higher concentrations of Iso to directly inhibit the stimulatory response. PDBu treatment produced similar effects on H2 histamine receptor-mediated ICa,L responses. In the presence of PDBu, higher concentrations of Iso inhibited the histamine stimulated ICa,L, and this effect was blocked by a selective beta1-AR antagonist. Higher concentrations of histamine also inhibited the Iso stimulated ICa,L, and this effect was blocked by a selective H2 receptor antagonist. The effects of PDBu were blocked by the PKC inhibitor bisindolylmaleimide I, and they were not mimicked by the inactive phorbol ester 4alpha-phorbol-12,13-didecanoate. The inhibitory effects of Iso and histamine were significantly reduced when Gi/o mediated responses were blocked with pertussis toxin. These results suggest that PKC promotes coupling of cardiac beta1-adrenergic and H2 histamine receptors to Gi/o mediated inhibitory responses.

Phorbol ester induces CYP2E1 in astrocytes, through a protein kinase C- and tyrosine kinase-dependent mechanism.

Cytochrome P450 2E1 (CYP2E1) is highly inducible in a subset of astrocytes in vivo following ischemic or mechanical injury and in vitro by lipopolysaccharide or interleukin-1beta. In the present study, phorbol-12,13-dibutyrate (PDBu) was found to induce catalytically active CYP2E1 more than fourfold in cortical glial cultures. Little induction was seen up to 12 h, and full effects only at 21-24 h of PDBu treatment. CYP2E1 expression in PDBu-treated cells was enriched in a subset of astrocytes. The protein kinase C inhibitors, staurosporine and calphostin C, and the tyrosine kinase inhibitor genistein, but not its inactive analogue daidzein, prevented the induction of CYP2E1 by PDBu. It is suggested that CYP2E1, together with interleukin-6 and ciliary neurotrophic factor, is part of a response of astrocytes to cellular stress elicited by, e.g. cerebral injury, cytokines or phorbol ester, and mediated in part through protein kinase C.

Conventional Protein Kinase C Mediates Phorbol-Dibutyrate-Induced Cytoskeletal Remodeling in A7r5 Smooth Muscle Cells

Phorbol dibutyrate (PDBu) induced the formation of podosome-like structures together with partial disassembly of actin stress fibers in A7r5 smooth muscle cells. These podosomes contained α-actinin, F-actin, and vinculin and exhibit a tubular, column-like structure arising perpendicularly from the bottom of PDBu-treated cells. The conventional protein kinase C (PKC) antagonist, GÖ6976, inhibited PDBu-induced cytoskeletal remodeling at 0.1 μM, whereas the novel PKC antagonist, rottlerin, was ineffective at 10 μM. PDBu induced the translocation of the conventional PKC-α but not the novel PKC-δ to the sites of podosome formation in A7r5 cells. Although partial disassembly of actin stress fibers was observed in both Y-27632- and PDBu-treated cells, focal adhesions were much reduced in number and size only in Y-27632-treated cells. Furthermore, PDBu restored focal adhesions in Y-27632-treated cells. Live video fluorescence microscopy of α-actinin GFP revealed a lag phase of about 20 min prior to the rapid formation and dynamic reorganization of podosomes during PDBu treatment. These findings suggest that conventional PKCs mediate PDBu-induced formation of dynamic podosome-like structures in A7r5 cells, and Rho-kinase is unlikely to be the underlying mechanism. The podosome columns could represent molecular scaffolds where PKC-α phosphorylates regulatory proteins necessary for Ca 2+ sensitization in smooth muscle cells.