Mary E. Reyland

Clinical management of salivary gland hypofunction and xerostomia in head-and-neck cancer patients: Successes and barriers

The most significant long-term complication of radiotherapy in the head-and-neck region is hyposalivation and its related complaints, particularily xerostomia. This review addresses the pathophysiology underlying irradiation damage to salivary gland tissue, the consequences of radiation injury, and issues contributing to the clinical management of salivary gland hypofunction and xerostomia. These include ways to (1) prevent or minimize radiation injury of salivary gland tissue, (2) manage radiation-induced hyposalivation and xerostomia, and (3) restore the function of salivary gland tissue damaged by radiotherapy.

Suppression of Apoptosis in the Protein Kinase Cδ Null Mouse in Vivo

Protein kinase C (PKC) δ is an essential regulator of mitochondrial dependent apoptosis in epithelial cells. We have used the PKCδ-/- mouse to ask if loss of PKCδ protects salivary glands against γ-irradiation-induced apoptosis in vivo and to explore the mechanism underlying protection from apoptosis. We show that γ-irradiation in vivo results in a robust induction of apoptosis in the parotid glands of wild type mice, whereas apoptosis is suppressed by greater than 60% in the parotid glands of PKCδ-/- mice. Primary parotid cells from PKCδ-/- mice are defective in mitochondrial dependent apoptosis as indicated by suppression of etoposide-induced cytochrome c release, poly(ADP-ribose) polymerase cleavage, and caspase-3 activation. Notably, apoptotic responsiveness can be restored by re-introduction of PKCδ by adenoviral transduction. Etoposide and γ-irradiation-induced activation of p53 is similar in primary parotid cells and parotid glands from PKCδ+/+ and PKCδ-/- mice, indicating that PKCδ functions downstream of the DNA damage response. In contrast, activation of the c-Jun amino-terminal kinase is reduced in primary parotid cells from PKCδ-/- cells and in parotid C5 cells, which express a dominant inhibitory mutant of PKCδ. Similarly, c-Jun amino-terminal kinase activation is suppressed in vivo in γ-irradiated parotid glands from PKCδ-/- mice. These studies indicate an essential role for PKCδ downstream of the p53 response and upstream of the c-Jun amino-terminal kinase activation in DNA damage-induced apoptosis in vivo and in vitro.

Induction of Apoptosis Is Driven by Nuclear Retention of Protein Kinase Cδ

Protein kinase Cδ (PKCδ) mediates apoptosis downstream of many apoptotic stimuli. Because of its ubiquitous expression, tight regulation of the proapoptotic function of PKCδ is critical for cell survival. Full-length PKCδ is found in all cells, whereas the catalytic fragment of PKCδ, generated by caspase cleavage, is only present in cells undergoing apoptosis. Here we show that full-length PKCδ transiently accumulates in the nucleus in response to etoposide and that nuclear translocation precedes caspase cleavage of PKCδ. Nuclear PKCδ is either cleaved by caspase 3, resulting in accumulation of the catalytic fragment in the nucleus, or rapidly exported by a Crm1-sensitive pathway, thereby assuring that sustained nuclear accumulation of PKCδ is coupled to caspase activation. Nuclear accumulation of PKCδ is necessary for caspase cleavage, as mutants of PKCδ that do not translocate to the nucleus are not cleaved. However, caspase cleavage of PKCδ per se is not required for apoptosis, as an uncleavable form of PKCδ induces apoptosis when retained in the nucleus by the addition of an SV-40 nuclear localization signal. Finally, we show that kinase negative full-length PKCδ does not translocate to the nucleus in apoptotic cells but instead inhibits apoptosis by blocking nuclear import of endogenous PKCδ. These studies demonstrate that generation of the PKCδ catalytic fragment is a critical step for commitment to apoptosis and that nuclear import and export of PKCδ plays a key role in regulating the survival/death pathway.

Suppression of cell migration by protein kinase Cdelta.

The ability of cancer cells to migrate is strongly correlated with malignant progression and metastasis. Survival signals that suppress apoptosis have also been linked to increased cell motility. We previously reported that suppression of protein kinase Cδ (PKCδ) provided survival signals in a rat fibroblast model system. These studies have been extended to human breast cancer cells with differential cell motilities and PKCδ levels. BT-549 cells, which lack detectable expression of PKCδ, migrate very efficiently, whereas MCF-7 cells, which express high levels of PKCδ, migrate very poorly. Ectopic expression of PKCδ suppressed cell migration in the BT-549 cells, and downregulation of PKCδ enhanced cell migration in the MCF-7 cells. Downregulation of PKCδ in the MCF-7 cells also led to increased secretion of the matrix metalloprotease MMP-9. The migration of mouse embryo fibroblasts (MEFs) from wild type and PKCδ knockout mice was also examined and MEFs from PKCδ knockout mice had a five-fold increase in cell migration relative to the wild-type MEFs. These data provide evidence that PKCδ suppresses cell migration in both human breast cancer cells and in primary mouse fibroblasts, and indicate that the loss of PKCδ in human cancers could contribute to both cell survival and metastasis.

TYROSINE PHOSPHORYLATION REGULATES NUCLEAR TRANSLOCATION OF PKCδ

PKCδ is essential for apoptosis, but regulation of the proapoptotic function of this ubiquitous kinase is not well understood. Nuclear translocation of PKCδ is necessary and sufficient to induce apoptosis and is mediated via a C-terminal bipartite nuclear localization sequence. However, PKCδ is found predominantly in the cytoplasm of nonapoptotic cells, and the apoptotic signal that activates its nuclear translocation is not known. We show that in salivary epithelial cells, phosphorylation at specific tyrosine residues in the N-terminal regulatory domain directs PKCδ to the nucleus where it induces apoptosis. Analysis of each tyrosine residue in PKCδ by site-directed mutagenesis identified two residues, Y64 and Y155, as essential for nuclear translocation. Suppression of apoptosis correlated with suppressed nuclear localization of the Y → F mutant proteins. Moreover, a phosphomimetic PKCδ Y64D/Y155D mutant accumulated in the nucleus in the absence of an apoptotic signal. Forced nuclear accumulation of PKCδ-Y64F and Y155F mutant proteins, by attachment of an SV40 nuclear localization sequence, fully reconstituted their ability to induce apoptosis, indicating that tyrosine phosphorylation per se is not required for apoptosis, but for targeting PKCδ to the nucleus. We propose that phosphorylation/dephosphorylation of PKCδ in the regulatory domain functions as a switch to promote cell survival or cell death.

Protein Kinase C δ Is a Downstream Effector of Oncogenic K-ras in Lung Tumors

Oncogenic activation of K-ras occurs commonly in non-small cell lung cancer (NSCLC), but strategies to therapeutically target this pathway have been challenging to develop. Information about downstream effectors of K-ras remains incomplete, and tractable targets are yet to be defined. In this study, we investigated the role of protein kinase C δ (PKCδ) in K-ras-dependent lung tumorigenesis by using a mouse carcinogen model and human NSCLC cells. The incidence of urethane-induced lung tumors was decreased by 69% in PKCδ-deficient knockout (δKO) mice compared with wild-type (δWT) mice. δKO tumors are smaller and showed reduced proliferation. DNA sequencing indicated that all δWT tumors had activating mutations in KRAS, whereas only 69% of δKO tumors did, suggesting that PKCδ acts as a tumor promoter downstream of oncogenic K-ras while acting as a tumor suppressor in other oncogenic contexts. Similar results were obtained in a panel of NSCLC cell lines with oncogenic K-ras but which differ in their dependence on K-ras for survival. RNA interference-mediated attenuation of PKCδ inhibited anchorage-independent growth, invasion, migration, and tumorigenesis in K-ras-dependent cells. These effects were associated with suppression of mitogen-activated protein kinase pathway activation. In contrast, PKCδ attenuation enhanced anchorage-independent growth, invasion, and migration in NSCLC cells that were either K-ras-independent or that had WT KRAS. Unexpectedly, our studies indicate that the function of PKCδ in tumor cells depends on a specific oncogenic context, as loss of PKCδ in NSCLC cells suppressed transformed growth only in cells dependent on oncogenic K-ras for proliferation and survival.

PKCαβγ- and PKCδ-dependent endocytosis of NBCe1-A and NBCe1-B in salivary parotid acinar cells

We examined membrane trafficking of NBCe1-A and NBCe1-B variants of the electrogenic Na(+)-HCO(3)(-) cotransporter (NBCe1) encoded by the SLC4A4 gene, using confocal fluorescent microscopy in rat parotid acinar cells (ParC5 and ParC10). We showed that yellow fluorescent protein (YFP)-tagged NBCe1-A and green fluorescent protein (GFP)-tagged NBCe1-B are colocalized with E-cadherin in the basolateral membrane (BLM) but not with the apical membrane marker zona occludens 1 (ZO-1). We inhibited constitutive recycling with monensin and W13 and detected that NBCe1-A and NBCe1-B accumulated in vesicles marked with the early endosomal marker early endosome antigen-1 (EEA1), with a parallel loss from the BLM. We observed that NBCe1-A and NBCe1-B undergo massive carbachol (CCh)-stimulated redistribution from the BLM into early endosomes. We showed that internalization of NBCe1-A and NBCe1-B was prevented by the general PKC inhibitor GF-109203X, the PKCalphabetagamma-specific inhibitor Gö-6976, and the PKCdelta-specific inhibitor rottlerin. We verified the involvement of PKCdelta by blocking CCh-induced internalization of NBCe1-A-cyan fluorescent protein (CFP) in cells transfected with dominant-negative kinase-dead (Lys376Arg) PKCdelta-GFP. Our data suggest that NBCe1-A and NBCe1-B undergo constitutive and CCh-stimulated endocytosis regulated by conventional PKCs (PKCalphabetagamma) and by novel PKCdelta in rat epithelial cells. To help develop a more complete model of the role of NBCe1 in parotid acinar cells we also investigated the initial phase of the secretory response to cholinergic agonist. In an Ussing chamber study we showed that inhibition of basolateral NBCe1 with 5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1H-indole-1-carboxamide (tenidap) significantly decreases an initial phase of luminal anion secretion measured as a transient short-circuit current (I(sc)) across ParC10 cell monolayers. Using trafficking and functional data we propose a model that describes a physiological role of NBC in salivary acinar cell secretion.

Protein kinase Cδ is required for ErbB2-driven mammary gland tumorigenesis and negatively correlates with prognosis in human breast cancer.

Protein kinase C δ (PKCδ) regulates apoptosis in the mammary gland, however, the functional contribution of PKCδ to the development or progression of breast cancer has yet to be determined. Meta-analysis of ErbB2-positive breast cancers shows increased PKCδ expression, and a negative correlation between PKCδ expression and prognosis. Here, we present in-vivo evidence that PKCδ is essential for the development of mammary gland tumors in a ErbB2-overexpressing transgenic mouse model, and in-vitro evidence that PKCδ is required for proliferative signaling downstream of the ErbB2 receptor. Mouse mammary tumor virus (MMTV)-ErbB2 mice lacking PKCδ (δKO) have increased tumor latency compared with MMTV-ErbB2 wild-type (δWT) mice, and the tumors show a dramatic decrease in Ki-67 staining. To explore the relationship between PKCδ and ErbB2-driven proliferation more directly, we used MCF-10A cells engineered to express a synthetic ligand-inducible form of the ErbB2 receptor. Depletion of PKCδ with short hairpin RNA inhibited ligand-induced growth in both two-dimensional (2D) (plastic) and three-dimensional (3D) (Matrigel) culture, and correlated with decreased phosphorylation of the ErbB2 receptor and reduced activation of Src and MAPK/ERK pathways. Similarly, in human breast cancer cell lines in which ErbB2 is overexpressed, depletion of PKCδ suppresses proliferation, Src and ERK activation. PKCδ appears to drive proliferation through the formation of an active ErbB2/PKCδ/Src signaling complex, as depletion of PKCδ disrupts association of Src with the ErbB2 receptor. Taken together, our studies present the first evidence that PKCδ is a critical regulator of ErbB2-mediated tumorigenesis, and suggest further investigation of PKCδ as a target in ErbB2-positive breast cancer.

Regulated Binding of Importin-α to Protein Kinase Cδ in Response to Apoptotic Signals Facilitates Nuclear Import

Background: Tyrosine phosphorylation regulates nuclear translocation of proapoptotic protein kinase C delta (PKCδ). Results: Tyrosine phosphorylation causes a conformational change that exposes the nuclear localization sequence, allowing binding of importin-α. Conclusion: Nuclear localization of PKCδ is regulated by access of importin-α to the nuclear localization sequence. Significance: Nuclear import of PKCδ, which induces apoptosis, is tightly regulated so as to prevent inappropriate cell death. PKCδ translocates into the nucleus in response to apoptotic agents and functions as a potent cell death signal. Cytoplasmic retention of PKCδ and its transport into the nucleus are essential for cell homeostasis, but how these processes are regulated is poorly understood. We show that PKCδ resides in the cytoplasm in a conformation that precludes binding of importin-α. A structural model of PKCδ in the inactive state suggests that the nuclear localization sequence (NLS) is prevented from binding to importin-α through intramolecular contacts between the C2 and catalytic domains. We have previously shown that PKCδ is phosphorylated on specific tyrosine residues in response to apoptotic agents. Here, we show that phosphorylation of PKCδ at Tyr-64 and Tyr-155 results in a conformational change that allows exposure of the NLS and binding of importin-α. In addition, Hsp90 binds to PKCδ with similar kinetics as importin-α and is required for the interaction of importin-α with the NLS. Finally, we elucidate a role for a conserved PPxxP motif, which overlaps the NLS, in nuclear exclusion of PKCδ. Mutagenesis of the conserved prolines to alanines enhanced importin-α binding to PKCδ and induced its nuclear import in resting cells. Thus, the PPxxP motif is important for maintaining a conformation that facilitates cytosplasmic retention of PKCδ. Taken together, this study establishes a novel mechanism that retains PKCδ in the cytoplasm of resting cells and regulates its nuclear import in response to apoptotic stimuli.

Electrogenic NBCe1 (SLC4A4), but not electroneutral NBCn1 (SLC4A7), cotransporter undergoes cholinergic-stimulated endocytosis in salivary ParC5 cells

Cholinergic agonists are major stimuli for fluid secretion in parotid acinar cells. Saliva bicarbonate is essential for maintaining oral health. Electrogenic and electroneutral Na(+)-HCO(3)(-) cotransporters (NBCe1 and NBCn1) are abundant in parotid glands. We previously reported that angiotensin regulates NBCe1 by endocytosis in Xenopus oocytes. Here, we studied cholinergic regulation of NBCe1 and NBCn1 membrane trafficking by confocal fluorescent microscopy and surface biotinylation in parotid epithelial cells. NBCe1 and NBCn1 colocalized with E-cadherin monoclonal antibody at the basolateral membrane (BLM) in polarized ParC5 cells. Inhibition of constitutive recycling with the carboxylic ionophore monensin or the calmodulin antagonist W-13 caused NBCe1 to accumulate in early endosomes with a parallel loss from the BLM, suggesting that NBCe1 is constitutively endocytosed. Carbachol and PMA likewise caused redistribution of NBCe1 from BLM to early endosomes. The PKC inhibitor, GF-109203X, blocked this redistribution, indicating a role for PKC. In contrast, BLM NBCn1 was not downregulated in parotid acinar cells treated with constitutive recycling inhibitors, cholinergic stimulators, or PMA. We likewise demonstrate striking differences in regulation of membrane trafficking of NBCe1 vs. NBCn1 in resting and stimulated cells. We speculate that endocytosis of NBCe1, which coincides with the transition to a steady-state phase of stimulated fluid secretion, could be a part of acinar cell adjustment to a continuous secretory response. Stable association of NBCn1 at the membrane may facilitate constitutive uptake of HCO(3)(-) across the BLM, thus supporting HCO(3)(-) luminal secretion and/or maintaining acid-base homeostasis in stimulated cells.