Phospholipase D Signaling and Extracellular Signal-Regulated Kinase-1 and -2 Phosphorylation (Activation) Are Required for Maximal Phorbol Ester-Induced Transglutaminase Activity, a Marker of Keratinocyte Differentiation

Abstract

Protein kinase C (PKC)-activating 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates phospholipase D (PLD) activity in primary mouse epidermal keratinocytes. PLD catalyzes the hydrolysis of phosphatidylcholine to yield phosphatidic acid (PA), which can be dephosphorylated to produce PKC-activating diacylglycerol. In the presence of small amounts of a primary alcohol, PLD can instead produce novel phosphatidylalcohols at the expense of PA and diacylglycerol. Here, we have demonstrated that inhibiting PLD signal generation with 1-butanol reduced TPA-stimulated transglutaminase activity, a marker of keratinocyte differentiation. On the other hand, the structurally related tertiary alcohol tert-butanol, which cannot be used by PLD, had no effect on TPA-induced transglutaminase activity. Since TPA activates all conventional and novel PKC isoforms directly, yet cannot overcome 1-butanol-mediated inhibition, this result suggests that PLD mediates its effects on transglutaminase activity (and keratinocyte differentiation) through an effector enzyme system distinct from the conventional or novel PKC isoenzymes. Data in the literature suggest that PA can recruit Raf-1 to the membrane, where it can be activated and initiate the mitogen-activated protein kinase cascade that culminates in activation of extracellular signal-regulated kinase (ERK)-1 and -2. Indeed, we found that inhibition of ERK-1/2 phosphorylation (activation) inhibited TPA-induced transglutaminase activity. However, inhibition of PLD-mediated signal generation had only a small effect on TPA-elicited ERK-1/2 phosphorylation (activation), whereas inhibition of ERK-1/2 did not affect PLD activation, suggesting that these two pathways likely operate largely in parallel. Thus, our results suggest the independent involvement of the PLD and ERK-1/2 pathways in mediating transglutaminase activity and keratinocyte differentiation.Phorbol esters, such as 12-O-tetradecanoyl phorbol 13-acetate (TPA), are known to promote the formation of tumors in epidermis initiated with carcinogens (for review, see Rubin, 2002). The mechanism of this tumor promotion is unknown, but the identification of protein kinase C (PKC) as the predominant phorbol ester binding protein in cells (for review, see Nishizuka, 1995) suggests an involvement of this enzyme family in the process. However, the acute effect of TPA both in vitro and in vivo is to induce keratinocyte differentiation (discussed in Bollag et al., 1993), a response seemingly inconsistent with its tumor-promoting action. The mechanism and downstream pathways activated in this biphasic TPA response are still at present unclear.As mentioned above, the primary target of TPA in cells is thought to be the PKC family. PKC isoenzymes can be divided into the classical members PKC-␣, -␤I and -␤II, and -␥, which are phospholipid-dependent, calcium-sensitive, and activated by phorbol ester, or the physiological activator diacylglycerol. The novel isoforms PKC-␦, -⑀, -, and -are also phospholipid-dependent and activated by phorb...

A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival

“…Although TG2 activity can be regulated by changes in intracellular [Ca 2+ ] there is growing evidence that TG activity can also be modulated by phosphorylation [7,8]. The broad spectrum PKC inhibitor Ro 31-8220 and the MEK1/2 inhibitor PD 98059 completely blocked CPA and adenosine-induced TG2 activity, suggesting prominent roles for PKC and ERK1/2.…”

“…The broad spectrum PKC inhibitor Ro 31-8220 and the MEK1/2 inhibitor PD 98059 completely blocked CPA and adenosine-induced TG2 activity, suggesting prominent roles for PKC and ERK1/2. Although ERK1/2 has been implicated in modulating the cross-linking activity of TG1 [8] there are no reports suggesting a role for this protein kinase in TG2 regulation.…”

HCN Pacemaker Channel Activation Is Controlled by Acidic Lipids Downstream of Diacylglycerol Kinase and Phospholipase A2

“…Generation of phosphatidic acid by phospholipase D does not contribute to the 4␤PMA response PA is readily generated not only from DAG via DGK, but also from cleavage of phosphatidylcholine by the action of the PKCactivated enzyme phospholipase D (PLD) (Butikofer et al, 1993;Bollag et al, 2005). We therefore asked whether blocking the action of PLD by 1-butanol could occlude the 4␤PMA facilitation, which would indicate a role for this mechanism of PA synthesis in the phorbol response.…”

Role of transglutaminase 2 in PAC1 receptor mediated protection against hypoxia-induced cell death and neurite outgrowth in differentiating N2a neuroblastoma cells

“…The transamidating activity of TG1 (keratinocyte transglutaminase) is enhanced by phorbol ester-induced stimulation of protein kinase C (PKC) and extracellular signal-regulated kinase 1/2 (ERK1/2; Bollag et al, 2005). These findings suggest that the activity of TG can be regulated by signaling pathways associated with GPCRs.…”