Lung disease and PKCs

Pharmacological Research 55 (2007) 545–559

Review

Lung disease and PKCs Edward C. Dempsey a,b,∗ , Carlyne D. Cool c,d , Cassana M. Littler a a

Cardiovascular Pulmonary Research Laboratory, University of Colorado Health Sciences Center, Denver, CO 80262, United States b Pulmonary and Critical Care Section, Medical Service, Denver VA Medical Center, Denver, CO 80220, United States c Department of Pathology, University of Colorado Health Sciences Center, Denver, CO 80262, United States d Department of Medicine, National Jewish Medical and Research Center, Denver, CO 80220, United States Accepted 16 April 2007

Abstract The lung offers a rich opportunity for development of therapeutic strategies focused on isozymes of protein kinase C (PKCs). PKCs are important in many cellular responses in the lung, and existing therapies for pulmonary disorders are inadequate. The lung poses unique challenges as it interfaces with air and blood, contains a pulmonary and systemic circulation, and consists of many cell types. Key structures are bronchial and pulmonary vessels, branching airways, and distal air sacs defined by alveolar walls containing capillaries and interstitial space. The cellular composition of each vessel, airway, and alveolar wall is heterogeneous. Injurious environmental stimuli signal through PKCs and cause a variety of disorders. Edema formation and pulmonary hypertension (PHTN) result from derangements in endothelial, smooth muscle (SM), and/or adventitial fibroblast cell phenotype. Asthma, chronic obstructive pulmonary disease (COPD), and lung cancer are characterized by distinctive pathological changes in airway epithelial, SM, and mucous-generating cells. Acute and chronic pneumonitis and fibrosis occur in the alveolar space and interstitium with type 2 pneumocytes and interstitial fibroblasts/myofibroblasts playing a prominent role. At each site, inflammatory, immune, and vascular progenitor cells contribute to the injury and repair process. Many strategies have been used to investigate PKCs in lung injury. Isolated organ preparations and whole animal studies are powerful approaches especially when genetically engineered mice are used. More analysis of PKC isozymes in normal and diseased human lung tissue and cells is needed to complement this work. Since opposing or counter-regulatory effects of selected PKCs in the same cell or tissue have been found, it may be desirable to target more than one PKC isozyme and potentially in different directions. Because multiple signaling pathways contribute to the key cellular responses important in lung biology, therapeutic strategies targeting PKCs may be more effective if combined with inhibitors of other pathways for additive or synergistic effect. Mechanisms that regulate PKC activity, including phosphorylation and interaction with isozyme-specific binding proteins, are also potential therapeutic targets. Key isotypes of PKC involved in lung pathophysiology are summarized and current and evolving therapeutic approaches to target them are identified. © 2007 Elsevier Ltd. All rights reserved. Keywords: Pulmonary vasculature; Airway; Alveolar; Interstitial; Injury; Inflammation; Cancer; Contraction; Proliferation; Apoptosis; Migration; PKC-alpha; PKC-beta; PKC-delta; PKC-epsilon; PKC-theta; PKC-eta; PKC-zeta; PKC-iota; Human; Knockout mice

Contents 1. 2. 3. 4. 5.

Introduction: the lung and its unique challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sites of injury: lung structure and cellular components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The link between injurious environmental stimuli and PKCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Strategies to implicate PKCs in lung cell injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PKCs in lung diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Vasculature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Airway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Alveolar wall/interstitium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Circulating cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗

546 546 546 547 550 550 551 552 553

Corresponding author at: Cardiovascular Pulmonary Research Laboratory, B-133, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Denver, CO 80262, United States. Tel.: +1 303 315 4483; fax: +1 303 315 4871. E-mail address: [email protected] (E.C. Dempsey). 1043-6618/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2007.04.010

546

6.

7.

E.C. Dempsey et al. / Pharmacological Research 55 (2007) 545–559

Therapeutic potential of targeting selected PKC isotypes in the lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Vasculature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Airway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Alveolar wall/interstitium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Circulating cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. Other concepts and relevant agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding thoughts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Find better experimental approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Explore lung-specific drug delivery strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Develop more accessible biomarkers of disease and drug activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4. Find ways of identifying patients with greatest likelihood of responding to PKC targeted therapy . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5. Show relevance of PKC biology to human lung disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6. Address still unanswered mechanistic questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7. Explore new angles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction: the lung and its unique challenges The lung offers a rich opportunity for development of therapeutic strategies focused on isozymes of protein kinase C (PKCs). PKCs are important in many cellular responses in the lung including permeability, contraction, migration, hypertrophy, proliferation, apoptosis, and secretion [1]. Existing therapies for most pulmonary diseases are inadequate. The lung interfaces with both air and blood. Therefore, drugs can be directly delivered to the lung by aerosol as well as oral and parenteral routes. The lung also poses unique challenges as it contains both a pulmonary and systemic circulation and consists of many different cell types. Expression patterns for PKCs and some of their regulatory features are cell-type specific making therapeutic targeting more difficult. 2. Sites of injury: lung structure and cellular components The key structures of the lung are branching airways, bronchial and pulmonary vessels, and distal air sacs defined by alveolar walls containing capillaries and interstitial space (Fig. 1A–C) [2]. Fig. 1A shows the major structures in normal human lung. Proximal pulmonary artery (PA) consists of (i) an endothelium, (ii) a media defined by an inner and outer elastic lamina, and (iii) an outer adventitia. Not demonstrated in this image are nearby veins and lymphatics. A small bronchial artery (i.e. vaso vasorum) is shown at the outer edge of the PA. Airways consist of (i) an epithelium with occasional mucous-secreting goblet cells, (ii) a medial layer, and (iii) an outer adventitia [3]. The yellow stain shows normal collagen deposition adjacent to large vessels and airways. In the upper left corner a few alveolar walls and underinflated spaces are shown. Fig. 1B and C demonstrate the simpler proximal and distal structures in mouse lung. There is scant adventitia around either PA or airway. Mice lack a bronchial circulation in the lung parenchyma. Their bronchial vasculature is abbreviated and ends at the level of the main bronchi. Murine alveolar spaces are only a fraction the size of human alveoli.

554 554 555 555 555 555 555 556 556 556 556 556 556 556 556 556

The cellular composition of each lung structure is heterogeneous. Vessels are comprised of endothelial, smooth muscle (SM), and adventitial fibroblast cells. Airways are made up of epithelial, mucous secreting goblet, airway SM, and fibroblast cells. Alveolar walls consist of type 1 and 2 pneumocytes, endothelial cells in fine capillaries, and interstitial fibroblasts and myofibroblasts. These cells interface with epithelial cells in terminal bronchi [3]. In addition to multiple resident cell types, circulating inflammatory, immune, and vascular progenitor cells traffic to the lung in response to injury. These circulatory cells regulate the magnitude of the inflammatory response and the pace and direction of repair. 3. The link between injurious environmental stimuli and PKCs Injurious environmental stimuli can access the lung through either the airways or the pulmonary and systemic circulations. The time course and intensity of responses by resident and circulating cells may be regulated by PKCs. A variety of pathological stimuli likely signal, at least in part, through PKCs (Table 1). These diverse agents are known to induce many different cellular responses like contraction, permeability, growth, and secretion that are known to be PKC-dependent. In some instances direct evidence supporting a role for PKC has been established; in other cases it has not, but likely would be if investigated. Exposure to hypoxic air at altitude, or low oxygen tension that develops following impaired diffusion of inhaled room air (with normal oxygen concentration) stimulates activation of PKCs in vascular cells followed by vasoconstriction and increased permeability [4–8]. Hyperoxia induces oxidant stress, which can activate (and inactivate) PKCs causing vasoconstriction, edema, and injury [9–12]. Bronchoconstriction and vasoconstriction in the lung cause shape change of airway and vascular cells, increased mechanical forces, and activation of PKCs [6,13,14]. Inhalation of cold air can induce bronchoconstriction and even vasoconstriction in the lungs of susceptible individuals. Transduction of this temperature signal likely activates/inactivates selected PKCs in airway and potentially vascular cells. Similarly, inhala-

E.C. Dempsey et al. / Pharmacological Research 55 (2007) 545–559

547

Table 1 Summary of potential exogenous stimuli for PKC isozymes in the lung Stimuli Oxygen tension Hypoxia Hyperoxia Mechanical forces Pressure Shear stress Inhaled irritants Cold air Oxidants Other noxious chemicals Inhaled dust Asbestos Silica Beryllium Inhaled allergens causing hypersensitivity pneumonitis (HSP) Specific forms of HSP and examples of allergens that trigger lung inflammation: farmer’s lung (thermophilic Actinomycetes or fungus in moldy hay), mushroom worker’s lung (mushroom spores), wine maker’s lung (Botrytis cincrea mold on grapes), hot tub lung (Mycobacterium avium complex), bird fancier’s lung (droppings, feathers, and serum proteins), malt worker’s disease (Aspergillus fumigatus or A. clavatus in moldy barley), wood dust pneumonitis (Alternaria sp. or Bacillus subtilis), and pesticide-induced (pyrethrum)

Fig. 1. Histological analysis of normal lung from adult human and wild type mouse. Pentachrome (for human) and trichrome (for mouse) staining were performed on formalin-fixed paraffin-embedded lung sections. (A) Pentachrome stain of human lung section. (B) Trichrome stained mouse lung tissue showing proximal PA and airway. (C) Trichrome stained mouse lung tissue showing alveolar walls, spaces, and distal vessel. Arrows point to specific structures in the lung: thick arrow – PA, triangular arrow – adjacent airway with epithelial lining; thin arrow – bronchial artery in adventitia (i.e. vaso vasorum). Panels B and C were reproduced with minor modification with permission from American Journal of Physiology [2].

tion of irritant, often pro-oxidant, chemicals can injure airway epithelium, trigger bronchoconstriction and even severe edema formation. Since PKCs are activated by tissue injury, oxidant stress, and change in cellular shape, they likely play an important role here as well.

Inhalation of a variety of occupational or hobby related dusts like asbestos and silica leads to a more slowly progressive inflammatory process culminating in lung fibrosis. Since PKCs have been implicated in growth, phenotypic modulation, and matrix deposition of fibroblasts, they likely contribute to the progression of these insidious lung disorders [15–19]. Finally, inhalation of a variety of allergens in the home and workplace can, in susceptible individuals, initiate an acute and then chronic inflammatory reaction. Histologically, this process (called ‘hypersensitivity pneumonitis’) is characterized by inflammation, granuloma formation with giant cells, and eventually fibrosis [20]. Many of the steps leading to this form of interstitial lung disease (ILD) are, at least in part, dependent on PKCs. This cascade includes initial recognition of the allergen, trafficking and activation of lymphocytes and monocytes, increased capillary permeability, activation and proliferation of interstitial fibroblasts, and matrix protein deposition [20]. 4. Strategies to implicate PKCs in lung cell injury The simplest strategy to investigate the role of PKC in the lung involves the application of cell permeable phorbol PKC activators (for additional details see Ref. [1]). The goal is either to directly mimic a relevant biological stimulus by selectively activating PKC or to use PKC activation to precondition or ‘prime’ the cell preparation to respond to another stimulus in a biologically relevant way. This approach was used to initially implicate PKC in the proliferative response of PA smooth muscle cells (SMCs) to hypoxia (Fig. 2A and B) [4]. PKC can

548

E.C. Dempsey et al. / Pharmacological Research 55 (2007) 545–559

Fig. 2. Implicating PKCs in hypoxic growth of pulmonary vascular cells. Synergistic proliferative response of PA SMC to PMA and hypoxia. Data are means ± S.E.; n = 5 replicate wells. (A) PMA-treated, but not untreated, quiescent PA SMC have increased [3 H] thymidine incorporation in response to moderate hypoxia. Inhibition of basal (no PMA) [3 H] thymidine incorporation noted with 3 and 0% O2 (*p < 0.05). Significant O2 concentration-dependent enhancement of [3 H] thymidine incorporation by PA SMC treated with 10 nM PMA and then exposed to mild-to-moderate hypoxia (10, 7, and 3% O2 ) shown above (**p < 0.05 compared with PMA-20% O2 -treated control). (B) PA SMC hypoxic proliferative response is dependent on priming concentration of PMA. Quiescent PA SMC were primed with 0.3 nM to 1 ␮M PMA and exposed to 20 or 3% O2 for 24 h. Under normoxic conditions, PMA stimulated an increase in [3 H] thymidine incorporation with a threshold at 1 nM and maximal effect at 10 nM (*p < 0.05). A further increase in [3 H] thymidine incorporation with exposure to 3% O2 was first detectable at 0.6 nM, and peaked at 10 nM. (**p < 0.05 compared with matched PMA-20% O2 treated group). Reproduced with permission from American Journal of Physiology [4].

then be further examined with broad pathway-specific inhibitors like chlerythrine, Ro-31-8220, calphostin-C, or sphingosine derivatives [21]. If PKC is important, the isozymes involved should be identified. If specific PKCs are not present, then they do not have a role in the lung disease of interest; but, if the method of detection is insensitive, then isotypes, which are in fact present, will be missed. Inflammation or injury may also induce expression of PKCs not initially detected. Western and, in selected studies, Northern blotting with isozyme-specific probes are used to detect expression of PKCs. The level of any one PKC isotype represents a balance between expression and degradation, a bal-

ance that may be altered in settings of cellular stress, injury, or transformation [21–24]. Relative amounts of the different PKCs cannot be determined easily as each probe has a different affinity. Determining which isoform is responsible for a change in phenotype usually requires further investigation. Identifying the individual PKCs activated in lung tissue or isolated cells in response to an environmental injury can be difficult. Traditionally, isozyme activation is detected by measuring intracellular translocation to membrane or cytoskeleton. Translocation to membrane can be detected by Western blotting, measurement of catalytic activity following subcellular fractionation, or immunostaining with confocal imaging of intact cells. Sensitivity of these assays is dependent on antibody affinity. Immunoprecipitation/kinase assays are also used to evaluate activity of individual PKC isozymes [25]. The phosphorylation state of PKCs is a critical determinant of activity [26–28]. Antibodies that detect the phosphorylated form of a few PKCs have been described [29]. A major concept to emerge is the importance of localization as a determinant of isozyme specificity [30–32]. Receptors for activated C-kinase (RACKs), caveolins, and other binding proteins contribute to an elaborate level of intracellular organization and compartmentalization for PKCs. The localization facilitates cross-talk between different signaling intermediates, targeted substrate phosphorylation, and regulation of catalytic activity. Expression of these binding proteins can be measured in lung tissue and cells and is important in normal and abnormal cellular function [33]. To further implicate individual isozymes in specific cell responses, several agonist and antagonist strategies with varying degrees of specificity have been developed. These techniques include pretreatment for 4–24 h with a high concentration of phorbol ester or bryostatin [21,34], application of inhibitors targeting either the catalytic or regulatory domain [35] or translocation itself [36,37], and the introduction of antisense RNA, dominant negative proteins, or, more recently, siRNA via transfection or viral (adeno or lenti) infection [38–40] (Fig. 3A–C) [34]. Complementary approaches should be used. When there are questions of inhibitor specificity and when cell-type specific effects have been observed, as is the case with phorbol ester-induced downregulation, the approach should be validated in the relevant cell system [21,34]. Inhibitors, like Go-6976, which target the catalytic domain, are competitive with ATP [35]. Specificity issues have been raised with these compounds (IC50 for purified PKC may be lower than for PKC in intact cells) but they are still in routine use. The putative PKC-␦ inhibitor, rottlerin, has also been shown to be less specific than originally thought [41], and therefore should be used with a complementary and more PKC-␦ specific approach. Myristolation allows introduction of pseudosubstrate peptides into viable cells [42]; similar strategies have been described to facilitate permeabilization of peptide translocation inhibitors [37,43]. Overexpression studies can also be used to explore potential roles of individual isozymes in cell function and complement antagonist strategies (Fig. 3D–F) [44]. These studies require complementary approaches, since an increase in one isozyme can alter the level of others [45,46].

E.C. Dempsey et al. / Pharmacological Research 55 (2007) 545–559

549

Fig. 3. Implicating individual PKCs in developmental and hypoxia-induced differences in growth responses of pulmonary vascular cells. Developmental difference: Ca2+ -dependent ␣ and ␤II isozymes of PKC contribute to the augmented growth of immature PA adventitial fibroblasts. (A) Go-6976 (3 ␮M; a more specific inhibitor of the Ca2+ -dependent isozymes of PKC than GF-109203X) inhibited serum-stimulated growth of fetal and neonatal, but not of adult, fibroblasts (like GF-109203X). Cell counts were performed on day 5. n = 4 replicate wells and *p < 0.05 compared with control cells for panels A and B. (B) Pretreatment with PMA (1 ␮M) for 24 h has the same selective antiproliferative effect as Go-6976 or GF-109203X. PMA (1 ␮M) was added to the cells on day 1, and then growth was assessed between days 2 and 5. (C) Differential susceptibility of PKC isozymes in PA adventitial fibroblasts to PMA-induced downregulation. Effect of pretreatment with PMA (1 ␮M) for 24 h on expression of isozymes in neonatal PA adventitial fibroblasts. There is selective degradation of the Ca2+ -dependent ␣ and ␤II isozymes of PKC in response to PMA (1 ␮M). PKC-␦ and -␧ are susceptible and PKC-␨ and -␮ are resistant to PMA-induced degradation. A shift in phosphorylation state of PKC-␮ in response to the high dose of PMA was also observed. Lanes marked with ‘–’ and ‘+’ represent lysates of cells pretreated with either vehicle (−) or 1 ␮M PMA (+). Hypoxic response: PKC-␨ overexpression attenuates DNA synthesis in vascular fibroblasts. (D) PKC-␨ expression is augmented in fibroblasts transfected with constitutively active PKC-␨ (Myr PKC-␨). Quiescent cells were transiently transfected with vector containing MyrPKC-␨ and empty vector (PCMV5). After 48 h of transfection, cells were exposed to either normoxia or hypoxia for 24 h and harvested with lysis buffer. (E) MyrPKC-␨ does not affect PKC-␫ expression in fibroblasts. (F) BrdU incorporation in hypoxic fibroblasts is attenuated by MyrPKC-␨. n = 4 replicate wells. *p < 0.05 compared with PCMV5 normoxic control. **p < 0.05 compared with the PCMV5 hypoxic group. Transfected fibroblasts were exposed to either normoxia or hypoxia in the presence of BrdU for 24 h. Panels A–C reproduced with minor modification with permission from the American Journal of Physiology [34]. Panels D–F reproduced with minor modification with permission from Molecular Biology of the Cell [44].

The most integrated way to investigate the role of PKCs in the lung involves isolated organ preparations and whole animal models including null (knockout) and transgenic mice [43,47,48] (Fig. 4A and B) [2]. If available, isozyme-specific inhibitors applied ex vivo or in vivo can help prove that the phenotype observed with a null mouse is due to the lack of the gene product and not the consequence of a developmental change. The

availability of conditional knockout models may also be helpful. The use of knockout mice to study intracellular kinases in the lung has been hindered by cellular heterogeneity. However, more options are now available to regulate PKC expression in specific cell populations (surfactant protein C promoter for the type 2 cell; SM-22 for SMCs, Tie-2 for endothelial cells, and CCtCre for epithelial cells) [49].

550

E.C. Dempsey et al. / Pharmacological Research 55 (2007) 545–559

Fig. 4. Implicating individual PKCs in complex acute and chronic hypoxic responses using isozyme-specific knockout mice. (A) Initial characterization of the model: expression of PKC isozymes in lungs from PKC-␧ wild type (+/+) and null (−/−) mice. n = 3 mice/genotype. Whole lung homogenates from PKC-␧ +/+ and −/− mice were subjected to SDS-PAGE and immunoblot analysis with PKC isozyme-specific (␧, ␣, ␤I, ␤II, ␥, ␦, ␩, ␪, ␨, ␮) antibodies. (B) Testing acute response to hypoxia: representative PA pressure tracings for isolated salt-perfused lungs from a PKC-␧ wild type (+/+) and null (−/−) mouse. Three maximal hypoxic (0% O2 ) challenges followed by one submaximal hypoxic (3% O2 ) challenge were performed. Hypoxic pulmonary vasoconstriction (HPV) was blunted in PKC-␧ null compared to +/+ mice. A 20 min period of equilibration was established before vascular responses were measured. HPV was expressed as mmHg above baseline. Figure reproduced with minor modification with permission from the American Journal of Physiology [2]. Chronic adaptive response to hypoxia is detailed elsewhere [59].

The most clinically relevant, albeit descriptive, approach to assessing the role of PKCs in lung disease is by direct measurement of expression, activity, and extent of activation in human tissue. Despite the precision targeting of knockout mice, they only partially model human susceptibility to disease. In addition to subtle differences in lung structure (Fig. 1A versus Fig. 1B and C), mice differ from humans in their responses to lipopolysaccharide (LPS) (more resistant to sepsis), hypoxia (more resistant to remodeling), carcinogens (probably more resistant to tumor formation and develop different cell types), and cigarette smoke (more resistant to developing emphysema). Unfortunately, few PKC-related studies on human lung have been reported [50]. It is difficult to access ‘normal’ lung tissue from non-smoking donors and disease specimens are typically obtained at the time of transplant and therefore are, by definition, end stage. The few gene array studies on human lung specimens reported have not found major perturbations in PKC isozyme expression [51,52]. However, since even modest changes in these major signaling intermediates can alter cell phenotype and much of the regulation of PKCs occurs at the protein level (phosphorylation, localization, etc.), gene array studies in humans may underestimate the importance of PKCs. 5. PKCs in lung diseases 5.1. Vasculature Injury, activation, and phenotypic switching of PA endothelial, SM, and adventitial fibroblast cells lead to vascular disorders like pulmonary edema and pulmonary hypertension (PHTN).

The endothelium provides the interface between the blood and extravascular tissue of the lungs and normally restricts movement of water and protein [53]. In normal lung, fluid and proteins are filtered from the circulation through gaps between capillary endothelial cells into the alveolar interstitial space and normally do not enter the alveoli because the alveolar epithelium is made of very tight junctions [54]. Two types of pulmonary edema occur in humans: cardiogenic and noncardiogenic [54]. Cardiogenic pulmonary edema is due to an increase in hydrostatic pressure in the pulmonary capillaries which leads to increased transvascular fluid filtration. In contrast, noncardiogenic edema results from endothelial injury and dysfunction. Alterations in endothelial cell morphology including changes in cell surface area, cell volume, and plasma membrane composition have been described [55]. Endothelial cells express many PKCs including ␣, ␤, ␦, ␧, ␩, ␪, and ␨ [10]. PKC activation by phorbol esters increases endothelial permeability by increasing contraction of microvessel endothelial cells and intercellular gap formation [53]. When extravascular water is increased in anesthetized rabbits, the distribution of PKC is shifted to the high-density fraction of the plasma membrane suggesting PKC may be an important modulator in noncardiogenic edema [55]. Mediators of noncardiogenic edema including thrombin, TNF-␣, and H2 O2 , induce PKCs [53]. Thrombin disrupts endothelium and is associated with intravascular fibrin and PMN sequestration. Thrombin also causes an increase in cytosolic [Ca2+ ] and activation of selected PKCs. Thrombin-induced PKC activation is associated with endothelial cell contraction and alterations of the cytoskeleton, both of which are important in endothelial barrier dysfunction [53]. Harrington et al. found that PKC-␣ overexpression decreases endothelial barrier func-

E.C. Dempsey et al. / Pharmacological Research 55 (2007) 545–559

tion by destabilizing adherens junction complexes, while PKC-␦ may enhance barrier function [56]. TNF-␣ is a mediator of sepsis syndrome causing increased pulmonary vascular resistance and noncardiogenic edema. TNF-␣ also activates PKC-␣ in PA endothelium [53]. Siflinger-Birnboim and Johnson suggest a model for TNF-␣-induced edema formation that begins with TNF-␣ causing activation of PKC-␣ in PA endothelium leading to stabilization of actin fibers. This stabilization impairs junctional proteins and cell–cell adherence and ultimately results in endothelial barrier dysfunction [53]. ROS are generated by PMNs and macrophages in lung injury. PKC inhibitors, including staurosporine, Go6976, H7, and calphostin C prevent ROS (H2 O2 )-induced endothelial barrier dysfunction, suggesting a role for selected PKCs in noncardiogenic edema formation [53]. Hypoxia is also an important cause of noncardiogenic edema. In humans, rapid ascent to altitude causes the development of pulmonary edema in susceptible individuals. Stelzner et al. found that hypoxia directly causes an increase in pulmonary vascular permeability in rats and increased permeability of bovine endothelial cell monolayers [8]. Hypoxia also inhibits alveolar fluid re-absorption and decreases Na, K-ATPase activity in alveolar epithelial cells [57]. Endocytosis of Na, KATPase molecules from the plasma membrane is mediated by mitochondria-generated ROS, and occurs simultaneously with activation of PKC-␨ and phosphorylation of the Na, K-ATPase ␣1 subunit. Chronic hypoxia is the most common cause of PHTN. Pulmonary hypertensive changes develop in response to sustained hypoxia-induced vasoconstriction [6]. Although this disorder is a major cause of disability for patients with chronic lung, heart, and sleep disorders, no treatments other than supplemental oxygen and supportive care are currently available. A much smaller group of patients have other forms of PHTN (idiopathic and associated with or due to drugs, viruses, liver disease, collagen vascular diseases, and ILD). Drugs (calcium channel blockers, prostacyclin, endothelin receptor antagonists, and sildenafil) are used to treat some of these patients. Usually, they offer only partial relief of the problem. PKC-based therapy would complement these agents. The role of endothelial cells in chronic hypoxic PHTN has focused mostly on control of vascular tone and permeability, areas where PKCs are important. Abnormal growth of apoptosis-resistant endothelial cells has been promoted as an important mechanism in idiopathic PHTN [58]. Little is known about the role of PKCs here. PA SMCs express many PKCs, and a growing body of evidence suggests that the Ca2+ -dependent PKCs (␣ and ␤) are particularly important in their hypoxic growth and ultimately the development of chronic hypoxic PHTN [1,4,5,7,21]. Growth of adventitial fibroblasts is also an important component of the structural changes observed in chronic PHTN. Developmental stage and hypoxic exposure are both important in regulating proliferation of adventitial fibroblasts [5,21,34]. Das et al. found that bovine PA adventitial fibroblasts express several PKCs including ␣, ␤I , ␤II , ␦, ␧, ␨, and ␮ [34]. A variety of complementary strategies were used to show that PKC-␣ and -␤II contribute to the augmented proliferative response of immature bovine PA adventitial fibroblasts (Fig. 3A–C) [34]. PKC plays an important role in proliferation

551

by activating downstream MAP kinases like ERK. They also found that decreased expression of PKC-␨ leads to persistent ERK1/2 phosphorylation and increased proliferation of fibroblasts exposed to hypoxia. Overexpression of PKC-␨ attenuates this response (Fig. 3D–F) [44]. Isolated organ preparations and knockout mice have been used to study the role of PKC-␧ in chronic PHTN. Importantly, no adaptive change in expression of other PKCs was found in this preparation (Fig. 4A) [1,2]. Persistent hypoxic pulmonary vasoconstriction (HPV) leads to vascular remodeling and fixed PHTN, and is blunted by PKC-␧ deletion [2]. With chronic hypoxia, decreased PKC-␧ provides relative protection from pulmonary vascular remodeling but not increased vasoreactivity [59]. Therefore, this therapeutic strategy could be useful in the acute setting, but would have to be combined with other vasodilatory agents to be used chronically. 5.2. Airway PKCs are important signaling intermediates in chronic airway diseases like asthma and chronic obstructive pulmonary disease (COPD). Asthma is characterized by reversible bronchospasm resulting from an exaggerated bronchoconstrictor response to various stimuli. COPD is a spectrum of smoking-related lung disorders that range from chronic bronchitis (thickening of airway walls with hypersecretion of mucous) to emphysema (progressive destruction of alveolar walls); a subset of these patients also have reversible bronchospasm (asthma-like spectrum). The mechanisms underlying increased airway reactivity are not fully understood, but bronchial inflammation is clearly important. PKCs have been implicated in airway inflammation, bronchospasm, and mucous production. Characteristic morphologic changes in asthma include inflammatory cell infiltration and edema of bronchial walls. Eosinophils are prominent as well as mast cells, basophils, macrophages, lymphocytes, plasma cells and neutrophils. Resident airway epithelial cells produce pro-inflammatory mediators under the regulation of PKC-␦ [60]. Increased PKC-␦ activity potentiates NF-␬B dependent pro-inflammatory cytokine production in human airway epithelial cells, while expression of a dominant negative PKC-␦ mutant has attenuating effects. In asthmatic airways the response to ␤2 -adrenoceptor (␤2 -AR) agonists is reduced and this may be in part due to inflammatory mediators, like cysteinyl-leukotrienes (cysteinyl-LTs). Cysteinyl-LTs cause SM contraction, proliferation, eosinophil recruitment into airways, increased microvascular permeability, and mucous hypersecretion. Selected leukotrienes (LTD4 ) induce ␤2 -AR desensitization in human airway SMC through a PKC-dependent mechanism [61]. Antigen stimulation of mast cells elicits release of pro-inflammatory mediators including histamine, cysteinyl-LTs, and Th2-associated cytokines and TNF-␣. Utilizing bone marrow derived mast cells from knockout mice, Leitges and co-workers have shown that PKC-␦, but not PKC-␧, is a negative regulator of antigen-induced mast cell degranulation [62,63]. Cho et al. defined a different role for PKC-␦ in mast cell degranulation in rat basophilic leukemia RBL-2H3 cells [64]. They found that activation of PKC-␦ leads

552

E.C. Dempsey et al. / Pharmacological Research 55 (2007) 545–559

to the degranulation of mast cells with subsequent cysteinyl-LT synthesis while acting as a downstream mediator of the small GTP binding protein, Rac1. The transformed cell line is likely responsible for the opposite role identified here. Further studies in a relevant mouse model were limited by lack of an available PKC-␦ specific antagonist (as neither rottlerin nor GF109203X are). One of the more striking morphological changes that occurs in asthma as well as in COPD is occlusion of bronchioles with mucous. Hypersecretion of mucous results from hypertrophy of submucosal mucous glands and increased numbers of goblet cells in bronchiolar epithelium. MUC5B and MUC5AC are prominent airway mucins present in secretions from submucosal glands and goblet cells. Hewson et al. found that PMA induced MUC5AC expression in a human respiratory epithelial cell line (NCI-H292), and this response was attenuated with the pan PKC inhibitor, calphostin [65]. In asthma, SM hypertrophy and hyperplasia occur in the bronchial wall at the level of large and medium airways, while in COPD these changes occur at the level of the small airways [66]. In human airway SMCs, PKC-␣, -␤I , -␦, -␧, -␮, -␥, and -␨ are found in the cytosol and -␤II in the membrane under basal conditions [67]. The pro-inflammatory neuropeptide bradykinin (BK) causes activation of PKC-␣, -␤I , -␦, and -␧ when applied to airway SMC. BK also induces COX-2 protein expression and PGE2 accumulation in human airway SMC through a PKC-␧ dependent mechanism. PKC-␣ is increased in lungs of patients with COPD and is thought to be important in airway SMC hypertrophy and proliferation [68]. PKC-␨ activity is also increased in proliferating human airway SMCs. Finally, enhanced activation of PKC-␤I and decreased activation of PKC-␦ have been demonstrated in proliferating airway SMCs from hyper-responsive Fischer rats [66]. Here, PKC-␦ has been identified as a negative regulator of airway SMC growth. Collectively, these studies demonstrate that multiple PKCs (␣, ␤I, ␦, ␧, ␪, and ␨) are important in the characteristic airway constriction, inflammation, mucus production, and cellular growth observed in asthma and COPD. However, their expression patterns and contributions to the pathogenesis of asthma and COPD are cell-type specific. PKCs are thought to play a key role in neoplastic transformation [45,69]. Increased PKC levels are observed in malignant tissues and inhibition of PKC prevents asbestos-induced c-fos and c-jun proto-oncogene expression in the lung [70]. Most primary lung tumors are thought to arise from bronchial epithelium. Some may also develop from resident stem cells in the airway wall [71]. Activation of the embryonic signaling hedgehog pathway occurs in airway epithelium during repair of acute injury as well as in small cell lung cancer (SCLC) [72]. Some SCLCs may arise from an airway epithelial progenitor cell that retains both hedgehog signaling and primitive features of pulmonary neuroendocrine differentiation [72]. PKC may be a viable target for inhibiting this signaling pathway [73]. PKC-␧ and other PKCs are important in activating NF-␬B-dependent transcription, and the pan PKC inhibitor Ro 31–8220 blocked this NF-␬B-dependent transcription in bronchial epithelial BEAS2B cells [73].

Many PKCs have been shown to be important in lung tumorigenesis. PKC-␣ is ubiquitously expressed and is consistently altered in human tumor cells [74]. Early on, overexpression of PKC-␣ was shown to induce a more malignant phenotype [45]. Wang et al. showed that PKC-␣ was overexpressed in the human lung carcinoma cell line LTEPa-2 and inhibition of this PKC decreased tumor formation in nude mice inoculated with the LTEPa-2 lung carcinoma cell line [75]. Targeting PKC-␣ with antisense oligonucleotide (ASO), LY900003, has shown promise as a chemotherapeutic strategy for non-small cell lung cancer (NSCLC), but more as a sensitizing agent combined with other chemotherapeutic drugs than as a single agent [74]. PKC-␤ may also be important in tumor cell growth and survival, and testing with the oral PKC-␤ inhibitor, enzastaurin, is underway [76]. Increased PKC-␦ expression/activation has been found in some studies to be important in cell survival as well as in resistance to chemotherapeutic drugs in SCLC and NSCLC models [77]. Others have reported a bryostatin-1 induced decrease in PKC␦ expression having attenuating effects on cell proliferation in NSCLC models [40]. PKC-␧ prevents human NSCLC lines from undergoing apoptosis and regulates susceptibility to etoposide [78]. Chemoresistance is the primary cause for treatment failure in advanced cancers, and PKC-␩ has been found to play a role in this process [79]. When an antisense PKC-␩ (ASO7) inhibitor was combined with either vincristine or paclitaxel, it became synergetic in activating caspase-3 and regulating apoptosis [79]. PKC-␫ also plays an important role in lung carcinogenesis, as it is required for the transformed growth of NSCLC cells. Expression of PKC-␫ is an indicator of prognosis independent of tumor stage [80]. Regala et al. suggest that PKC-␫ is an oncogene and may be an important target for treatment of NSCLC [80]. 5.3. Alveolar wall/interstitium ILDs are a group of disorders characterized by interstitial inflammation and varying amounts of fibrosis. They can result from environmental exposures, be associated with systemic diseases, or be idiopathic [20,81]. ILDs are triggered by injury from inhalation of occupational dusts (silicosis, asbestosis), allergic reactions to inhaled allergens (hypersensitivity pneumonitis), collagen vascular diseases (rheumatoid arthritis, systemic lupus erythematosus, scleroderma), drugs (amiodarone, chemotherapeutic agents), infection (bronchiolitis, organizing pneumonia), and even recurrent episodes of congestive heart failure. The resultant lung injury is characterized by necrosis of type I pneumocytes, proliferation of type II pneumocytes, interstitial edema, accumulation of inflammatory cells and interstitial fibroblasts, as well as collagen and fibronectin synthesis [82]. During the acute phase, alveolar filling may occur to varying degrees (so called “edema formation”). When there is persistent or recurring injury and inflammation, cellular infiltration continues. Interstitial fibroblasts become activated, proliferate, and undergo phenotypic switching to myofibroblasts. Matrix protein (especially collagen) deposition accelerates, fibrosis results, and there is loss of lung function [83]. A few patients respond to removal of an identifiable trigger. Steroid therapy may also be useful for a small subgroup of patients with active inflammation. The

E.C. Dempsey et al. / Pharmacological Research 55 (2007) 545–559

majority, however, have not been shown to benefit from steroids or other immunosuppressive agents. PKC is a key regulator of fibrosis in human pulmonary interstitial fibroblasts. At least three PKCs are expressed in interstitial fibroblasts including PKC-␣, -␦, and -␧ [17]. PKC-␣ contributes bidirectionally (depending on context) to the regulation of collagen expression in human lung interstitial fibroblasts. Activation of PKC-␣ causes decreased collagen expression via the extracellular signal-regulated kinase kinase (MEK)/ERK signaling cascade, a response that is opposed by PKC-␧ [18]. The model proposed is that PKC-␣ activation leads to increased calveolin-1 expression in lung interstitial fibroblasts, and calveolin-1 modulates signal transduction by linking kinases together such as PKC to MEK/ERK with inhibition of their catalytic activity [18]. However, in a different setting PKC-␣ activation by a chemokine, CCL18, causes an increase in collagen production [17]. CCL18 is a calcium, PKC, and ERK dependent transcriptional activator of collagen and directly acts on cultured pulmonary interstitial fibroblasts to upregulate collagen production. CCL18 is increased in patients with scleroderma, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis, and sarcoidosis [17]. In interstitial fibroblasts, transforming growth factor-␤ (TGF␤) promotes synthesis and deposition of the extracellular matrix including collagen and may also alter the balance of matrix metalloproteinases [19]. TGF-␤ induces synthesis of collagen by phosphorylating Smad proteins, which then translocate to the nucleus and regulate transcriptional responses of genes. In a variety of cells including human pulmonary interstitial fibroblasts, TGF-␤-mediated activation of genes has been found to require PKC-␦ in addition to the Smad proteins. Interleukin7 (IL-7) negatively regulates collagen synthesis by inhibiting TGF-␤ signaling including PKC-␦ activity [19]. PKC-␧ is also important in upregulating collagen expression in normal human lung interstitial fibroblasts but via a different signaling cascade [18]. In these cells, activation of PKC-␧ leads sequentially to mitogen-activated protein kinase (MAPK) or MEK and ERK activation and enhanced collagen expression [18]. Collectively, these studies suggest that PKC-␣, -␦ and -␧ are likely important in the pathogenesis of chronic pulmonary fibrotic diseases. 5.4. Circulating cells Circulating inflammatory, immune, and vascular progenitor cells travel to the lung in response to various forms of injury including infection, asbestos, and cigarette smoke. Inflammatory and immune cells contribute to the injury and repair process. PKCs are important intermediates in the inflammatory response. IL-1 and interferon-␥ (IFN-␥) are important in modulating acute and chronic airway inflammation by increasing the release of arachidonic acid (AA) from epithelial cells [84]. IL-1 upregulates adhesion molecules, facilitates the migration of inflammatory cells into the airway wall and activates profibrotic mechanisms in the subepithelium. IFN-␥ inhibits Th-2 cell proliferation and has a pro-inflammatory effect by upregulating adhesion molecules and other cytokines [84]. PKC

553

is important in mediating pro-inflammatory cytokine effects by phosphorylating cytosolic PLA2 leading to release of AA from phospholipids with subsequent production of bioactive eicosanoids in activated cells [84]. Another pathway important in the regulation of inflammation is the complement-derived anaphylatoxin C5a pathway. The C5a receptor (C5aR) is constitutively expressed on human bronchial epithelial cells and these cells respond to C5a by releasing IL8. Cigarette smoke extract heightens C5aR responsiveness to C5a in these human bronchial epithelial cells by increasing the number of cells that express C5aR [85]. PKC is an important signaling component linked to C5aR in human neutrophils. Cigarette smoke extract activates PKC in other cell types including monocytes, endothelial cells, and lung carcinoma cells. Injury by cigarette smoke extract facilitates C5a-stimulated IL-6 and IL-8 release through a PKC-mediated pathway and leads to pro-inflammatory cytokine production [85]. Selected PKCs are activated by LPS, leading to the production of the pro-inflammatory cytokines TNF-␣, IL-1␤, and IL-6 [86]. Kontny et al. initially used the PKC inhibitor calphostin-C to implicate the PKC pathway in the synthesis of pro-inflammatory cytokines. They then correlated the translocation/activation of PKCs with pro-inflammatory cytokine production, and concluded that PKC-␣, -␤II , -␦, and -␧ isoforms may be important in the induction of cytokines in human monocytes cultured in vitro [86]. Thrombin mediates inflammatory and tissue-repair responses associated with vascular injury. Thrombin is also chemotactic for monocytes and is mitogenic for lymphocytes and mesenchymal cells. Thrombin causes an increase in cytosolic [Ca2+ ] and activation of selected PKCs [53]. After vascular injury, thrombin induces IL-6 and IL-8 in endothelial cells and fibroblasts leading to recruitment and activation of neutrophils within the local environment. Many inflammatory mediators are also capable of modulating functions of parenchymal cells involved in tissue repair. PGD2 is one example of this concept; it is a product of the AA pathway and is capable not only of bronchoconstriction, but it also can mediate effects on vessels and platelets. PGD2 is a product of dendritic cells, Th2 lymphocytes, and platelets. Both mast cells and Th2-derived mediators modulate fibroblast functions important in repair and remodeling in asthma [87]. PGD2 plays a role in remodeling and repair by reorganizing the extracellular matrix in vitro. PKC has been found to be involved in this repair process. By using a combination of inhibitor strategies, calcium-independent PKCs (such as PKC-␧) have been implicated in blocking PGD2 -induced contraction of collagen gels. These findings suggest that PKC may be an important mediator in tissue repair by increasing the ability of fibroblasts to contract their surrounding matrix [87]. Immune cells such as cytotoxic CD8+ T cells and T helper (Th) 1 cells are important to mounting an effective immune response against antigens such as viral infections. PKC-␪ is a key signaling molecule downstream of the T cell receptor and is important in Th2 cell differentiation and function. PKC-␪ is also involved in the activation of CD8+ cells in the antiviral response in vitro [88]. Because of its restricted expression and important

554

E.C. Dempsey et al. / Pharmacological Research 55 (2007) 545–559

role in inflammation, PKC-␪ is a potential therapeutic target for multiple lung disorders. Research on vascular progenitor cells is also challenging the dogma that proliferation and migration of resident cells are the only important contributors to the pathogenesis of acute and chronic lung diseases [89]. Davie et al. found that bone marrow (BM)-derived cells, including circulating endothelial, SM, and fibrocyte progenitor cells, may contribute to vascular remodeling in response to hypoxia [90]. They also observed that circulating mononuclear cells isolated from neonatal calves exposed to hypoxia differentiate into endothelial and SMC phenotypes. Characterization of circulating progenitor cells in tracheal and alveolar epithelium has also recently been undertaken [89]. In one study, mice with elastase-induced emphysema were treated with agents to mobilize BM-derived progenitor cells. An increase in BM-derived cell number in the alveoli of the emphysematous mice and lung regeneration was found [91]. Yamada et al. have reported a correlation between inflammatory disease processes such as bacterial pneumonia in humans and a rapid release of endothelial progenitor cells into the circulation [92]. These studies highlight the likely direction of therapeutic approaches to lung repair following pulmonary inflammation [93]. PKCs are almost certainly involved in the trafficking, proliferation, and differentiation of these progenitor cells. PKCs are known to be important in the regulation of hematopoietic cell proliferation and differentiation [94] and have been implicated in the differentiation of circulating progenitor cells to cardiomyogenic cells in the heart [95]. 6. Therapeutic potential of targeting selected PKC isotypes in the lung 6.1. Vasculature Efforts to target PKC as a treatment for lung disease actually began many years ago with studies of heparin. Heparin was found to attenuate chronic hypoxic PHTN in rodents by mechanisms independent of its anticoagulant effects [96]. This compound also decreased bronchoconstriction in asthma [97] and attenuated lung injury [98]. Susceptibility to the antipro-

liferative effects of heparin in the systemic circulation was subsequently shown to be dependent, at least in part, on PKCs [99–101]. Immature pulmonary vascular cells, isolated at a developmental stage when susceptibility to vascular injury is enhanced, were found to be particularly sensitive to heparin [102]. Classical PKCs were then implicated in both the heightened proliferative potential and the increased susceptibility to heparin of these SMCs and fibroblasts [5,21,34,102]. Obviously, heparin has many biological effects, but this link to PKCs provided part of a therapeutic rationale for further testing in a number of clinically relevant settings. Unfortunately, the chronic use of heparin has been limited by risk of osteoporosis. Other agents that target the classical PKC isozymes (␣ and ␤ in the lung) would also likely be efficacious in chronic forms of PHTN based on extensive in vitro testing in vascular cells (Table 2). However, the ability of these agents or relevant knockout mice to attenuate established PHTN is unknown. PKC-␦ agonists and antagonists have promise but may require cell specific targeting. While increased apoptosis in SMCs and fibroblasts could prevent medial and adventitial thickening, apoptosis of endothelial cells could cause vessel rarification (or dropout) and increased PHTN. Mochly-Rosen’s group has demonstrated the feasibility of selectively activating individual PKCs on a chronic basis for therapeutic benefit [30]. This could be useful for PKC-␦ in airway disorders and PKC-␨ in PHTN [44,62,66]. The PKC-␨ strategy might have to be directed to fibroblasts selectively since activation of this isotype in endothelial cells could increase permeability and worsen PHTN [57]. Decreased PKC-␧ blunts HPV, an autoregulatory mechanism that when sustained leads to fixed PHTN [2]. Vasodilatory agents that block this response typically have therapeutic effects in PHTN (calcium channel blockers, NO, prostacyclin, PDE4 and rho kinase inhibitors). However, based on in vivo chronic hypoxia studies it appears that PKC-␧ inhibition would require a second complementary vasodilatory strategy to be efficacious as the deletion of PKC-␧ alone has protective effects on vascular remodeling but not on tone [59]. Based on the work of Malik and co-workers, PKC-␤ inhibition may prevent edema formation in a variety of settings [103]. Interestingly, this group has recently implicated a number of additional isotypes in the regulation of permeability raising the

Table 2 Summary of potential PKC targets in various lung diseases Sitea

Disorders

Potential PKC targets goal Increased

Decreased

Reference

Vasculature

Pulmonary edema Pulmonary hypertension

␦ ␨

␣, ␤, ␧, ␨ ␣, ␤, ␧

[53,56,57] [2,4,5,7,21,34,44,59,102]

Airway

Asthma COPD Cancer

␦ ␦ ␦

␣, ␤, ␦, ␧, ␨ ␣, ␤, ␦, ␪, ␨ ␣, ␤, ␦, ␧, ␥, ␩, ␫, ␪, ␨

[60,62–64,66,67] [65,66,68] [69,70,74,76–80]

Alveolar wall/interstitium

Interstitial pneumonia Pulmonary fibrosis

␣

␣, ␦, ␧

[15–19]

␣, ␤, ␦, ␧, ␥, ␪

[87,88,105]

Circulating cells a

All of the above

Circulating cells (including inflammatory, immune and vascular progenitor cells) are involved at all sites in all the listed disorders and regulate at least in part the magnitude of the inflammatory response and extent of tissue injury seen and the timing of repair.

E.C. Dempsey et al. / Pharmacological Research 55 (2007) 545–559

possibility that combinational therapy targeting PKC-␣, -␧, and -␨, in addition to PKC-␤, may be most effective. 6.2. Airway PKC inhibitors suppress inflammation and NF-␬B activation and, therefore, could be efficacious in asthma [73]. Strategies to decrease inflammatory cell recruitment, suppress mucous production, and limit oxidant stress and protease activation in COPD could be helpful. Since PKC inhibitors decrease these endpoints, they may also be useful in this setting. Combinational strategies targeting several isotypes including ␣, ␤, ␦, ␪, and ␨ would probably be most effective (Table 2). Activation/overexpression and inhibition of PKC-␦ will require careful study, as both strategies may also be helpful in some settings [60,62,66]. At the present time two thirds of patients are incurable at the time of initial presentation with lung cancer. Therefore, more effective chemotherapeutic drugs are needed. Inhibition of PKC-␣ decreases growth of lung cancer cells and the multidrug resistance gene expression is dependent on this isotype, so initial therapeutic strategies were concentrated on this isozyme [74]. Single agent therapy using antisense (ISIS 3521) was not as effective as anticipated; therefore, the focus has shifted to other preparations and combinational therapy. More recent reports have implicated additional isotypes of PKC in the growth and metastatic potential of lung cancer cells and suggest combinational therapy may be more effective. Other isotypes implicated include ␤, ␦, ␧, ␥, ␩, ␫, ␪, and ␨. Initial evaluation of single isozyme antagonists is underway for at least a few of these PKCs including ␤, ␩ and ␫ [76,79,80]. Single agents (targeting PKC␣ or others) may still play a role in combination with either radiation therapy or other classes of chemotherapeutic drugs. 6.3. Alveolar wall/interstitium Strategies that decrease permeability, suppress inflammatory cell recruitment and cytokine release, block myofibroblast phenotypic conversion, and promote matrix protein degradation are most likely to be successful. Many agents have been tested before, but only removal of the inciting agent and, in a few settings, steroid therapy are helpful, and then only if initiated before extensive fibrosis develops. There is some evidence that decreasing or inhibiting PKC-␦ could have therapeutic benefit [19]. PKC-␧ inhibition may also block development of the myofibroblast phenotype [18]. 6.4. Circulating cells PKC antagonists with anti-inflammatory effects would likely be efficacious in lung disorders like asthma where inflammatory and/or immune cell recruitment is important. Several years ago, Nixon et al. demonstrated potent attenuating effects of selected bis(indolyl)-maleimide derivatives [104]. More recent literature also suggests drugs that inhibit classical PKCs would likely be effective here [105]. The potential benefits of targeting PKC␪ have been emphasized [88]. The expression of this isotype is restricted to selected inflammatory cells, so antagonist strategies

555

would likely blunt injury and inflammation. Since inflammatory cells are now thought to play such an important role in essentially all lung disorders, this approach could have efficacy in many settings. 6.5. Other concepts and relevant agents For several lung disorders, multiple PKCs have been implicated and successful intervention may require targeting of more than one isotype and/or a bidirectional approach (i.e. inhibit one and stimulate another). Another important concept to consider is the time course or stage of the disorder when treatment would be initiated. In airway disease and alveolar wall/interstitial processes some isozymes, like PKC-␣ and -␦, may play different roles at different stages. Thus, precise staging may be needed to determine whether upregulation or downregulation of the targeted PKC is likely to be helpful. There may be a role for well-tolerated drugs like tamoxifen, bryostatin, and related derivatives for treatment of nonmalignant lung disorders [106,107]. Because these agents have been extensively tested in humans in clinical cancer trials, their use could be accelerated in new areas. While these type of drugs should be tested alone initially, they may also have permissive, priming, or synergistic effects on target cells or tissues, making animal models and ultimately patients more responsive to other PKC-dependent or -independent therapeutic strategies. 7. Concluding thoughts This review has focused on the importance of PKCs in lung health and disease and the feasibility of selecting PKC isozymes as therapeutic targets [43,108,109]. Advances in PKC signaling have made it possible to investigate this remarkably complex enzyme family in vitro and in vivo with greater precision than in the past. Genetically engineered mice in which individual PKCs have been selectively manipulated offer a particularly powerful approach to identify viable therapeutic strategies [47,110]. More analysis of PKCs in normal and diseased human lung tissue and cells is needed to complement work in mice, as there are important anatomical differences in the lungs of the two species and limited information exists about PKCs in human lung [3]. Opposing or counter regulatory effects of selected PKCs have been found in the same cell or tissue preparation; therefore it may be desirable to target more than one PKC isozyme and potentially in different directions [30–32]. Since multiple signaling pathways contribute to many of the key cellular responses important in lung biology, it seems likely that therapeutic strategies targeting PKCs will be more effective if combined with inhibitors of other pathways for additive or synergistic effect [111]. Besides the PKCs themselves, there are a number of proteins involved in the regulation of the individual isotypes that show promise as potential therapeutic targets. Therefore, mechanisms that regulate PKC activity including phosphorylation (PDK-1), de-phosphorylation (selected phosphatases) and targeted translocation by isozyme-specific binding proteins (RACKs, caveolin, annexin) could also be potential targets for therapeutic intervention [26–28,30].

556

E.C. Dempsey et al. / Pharmacological Research 55 (2007) 545–559

Immediate challenges in the pulmonary field include. 7.1. Find better experimental approaches More efficient and selective ways to apply isozyme-specific antagonist strategies to primary cultures of epithelial, endothelial, SM, and fibroblast cells that are known to be heterogeneous and to isolated lung preparations and whole animal models are needed. 7.2. Explore lung-specific drug delivery strategies The lung offers the airway as an alternate approach for drug delivery. The advantage is the ability to potentially achieve higher local drug concentrations in the area of interest and avoid systemic toxicity. However, aerosol formulations also require a different series of safety and efficacy tests, which can delay clinical trials. Airway directed gene therapy has shown potential but viral vector-induced inflammation has been an issue.

injury and fibrosis. Targeting key effectors of inflammation may be more effective than other approaches. Regression is an understudied part of the disease process but is essential to the success of any therapeutic strategy. Therefore, there needs to be more emphasis on the role of PKCs in regression of disease. Acknowledgements We thank Dr. Mary Reyland for a critical review of this manuscript; Dr. Mita Das for assistance with Fig. 3; Drs. York Miller, Peter Hensen, and John Weil for helpful comments. ECD is supported by NHLBI PPG #HL14985, RO-1 #HL078927, a State of Colorado Biotechnology Discovery grant, and the Veterans Administration (VA). The work described was also supported by a VA Merit Review grant. CML was supported by a NIDDK 5 T35 DK07496-18 Short-Term Training in Health Professional School grant and Achievement Rewards for College Scientists Scholarship Fund. References

7.3. Develop more accessible biomarkers of disease and drug activity More accessible/less invasive ways of monitoring disease activity and drug levels and response to therapy are needed. This may involve analysis of changes in gene expression in circulating cells if they mirror disease activity in the lung. 7.4. Find ways of identifying patients with greatest likelihood of responding to PKC targeted therapy To figure out which subgroup will respond best will require greater use of proteomic and gene array strategies and analysis of more accessible cell samples than the lung provides [112]. 7.5. Show relevance of PKC biology to human lung disease More analysis of PKCs and related factors is needed in difficult to access normal and diseased human lung. Funding of multi-center tissue banking cores should help. Laser capture should help with the problem of heterogeneity in the analysis of human tissues. 7.6. Address still unanswered mechanistic questions What effect do relevant forms of cellular stress like hypoxia, hyperoxia, shear stress, and cigarette smoke have on expression and function of relevant PKC kinases (PDK-1), phosphatases, and binding proteins? Do changes in levels of PKCs occur under conditions of cellular stress and do they contribute to change in phenotype and by what mechanism? 7.7. Explore new angles Inflammation is a common thread among lung diseases like asthma, COPD, PHTN, and acute and chronic forms of lung

[1] Dempsey EC, Newton AC, Mochly-Rosen D, Fields AP, Reyland ME, Insel PA, et al. Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol 2000;279:L429– 38. [2] Littler CM, Morris Jr KG, Fagan KA, McMurtry IF, Messing RO, Dempsey EC. Protein kinase C-epsilon-null mice have decreased hypoxic pulmonary vasoconstriction. Am J Physiol Heart Circ Physiol 2003;284:H1321–31. [3] Maitra A, Kumar V. The lung and the upper respiratory tract. In: Kumar V, Cotran RS, Robbins SL, editors. Robbins basic pathology. Philadelphia: Saunders; 2003. p. 453–508. [4] Dempsey EC, Mcmurtry IF, O’Brien RF. Protein kinase C activation allows pulmonary artery smooth muscle cells to proliferate to hypoxia. Am J Physiol Lung Cell Mol Physiol 1991;260:L136–45. [5] Dempsey EC, Badesch DB, Dobyns EL, Stenmark KR. Enhanced growth capacity of neonatal pulmonary artery smooth muscle cells in vitro: dependence on cell size, time from birth, insulin-like growth factor I, and auto-activation of protein kinase C. J Cell Physiol 1994;160:469–81. [6] Dempsey EC, Stevens T, Durmowicz AG, Stenmark KR. Hypoxiainduced changes in contraction, growth, and matrix synthtic properties of pulmonary vascular cells. In: Haddad G, Lister G, editors. Tissue oxygen deprivation: from molecular to integrated function. New York: M. Dekker; 1996. p. 225–74. [7] Dempsey EC, Frid MG, Aldashev AA, Das M, Stenmark KR. Heterogeneity in the proliferative response of bovine pulmonary artery smooth muscle cells to mitogens and hypoxia: importance of protein kinase C. Can J Physiol Pharmacol 1997;75:936–44. [8] Stelzner TJ, O’Brien RF, Sato K, Weil JV. Hypoxia-induced increases in pulmonary transvascular protein escape in rats. Modulation by glucocorticoids. J Clin Invest 1988;82:1840–7. [9] Das KC, Pahl PM, Guo XL, White CW. Induction of peroxiredoxin gene expression by oxygen in lungs of newborn primates. Am J Respir Cell Mol Biol 2001;25:226–32. [10] Rahman A, Bando M, Kefer J, Anwar KN, Malik AB. Protein kinase C-activated oxidant generation in endothelial cells signals intercellular adhesion molecule-1 gene transcription. Mol Pharmacol 1999;55:575– 83. [11] Frey RS, Gao X, Javaid K, Siddiqui SS, Rahman A, Malik AB. Phosphatidylinositol 3-kinase gamma signaling through protein kinase Czeta induces NADPH oxidase-mediated oxidant generation and NF-kappaB activation in endothelial cells. J Biol Chem 2006;281:16128–38. [12] Mehta D, Rahman A, Malik AB. Protein kinase C-alpha signals rho-guanine nucleotide dissociation inhibitor phosphorylation and rho

E.C. Dempsey et al. / Pharmacological Research 55 (2007) 545–559

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20] [21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30] [31]

[32]

activation and regulates the endothelial cell barrier function. J Biol Chem 2001;276:22614–20. Komuro I, Katoh Y, Kaida T, Shibazaki Y, Kurabayashi M, Hoh E, et al. Mechanical loading stimulates cell hypertrophy and specific gene expression in cultured rat cardiac myocytes. Possible role of protein kinase C activation. J Biol Chem 1991;266:1265–8. Wang L, Liu HW, Mcneill KD, Stelmack G, Scott JE, Halayko AJ. Mechanical strain inhibits airway smooth muscle gene transcription via protein kinase C signaling. Am J Respir Cell Mol Biol 2004;31:54–61. Lounsbury KM, Stern M, Taatjes D, Jaken S, Mossman BT. Increased localization and substrate activation of protein kinase C delta in lung epithelial cells following exposure to asbestos. Am J Pathol 2002;160:1991–2000. Ding M, Huang C, Lu Y, Bowman L, Castranova V, Vallyathan V. Involvement of protein kinase C in crystalline silica-induced activation of the MAP kinase and AP-1 pathway. Am J Physiol Lung Cell Mol Physiol 2006;290:L291–7. Luzina IG, Highsmith K, Pochetuhen K, Nacu N, Rao JN, Atamas SP. PKCalpha mediates CCL18-stimulated collagen production in pulmonary fibroblasts. Am J Respir Cell Mol Biol 2006;35:298–305. Tourkina E, Gooz P, Pannu J, Bonner M, Scholz D, Hacker S, et al. Opposing effects of protein kinase Calpha and protein kinase Cepsilon on collagen expression by human lung fibroblasts are mediated via MEK/ERK and caveolin-1 signaling. J Biol Chem 2005;280:13879– 87. Zhang L, Keane MP, Zhu LX, Sharma S, Rozengurt E, Strieter RM, et al. Interleukin-7 and transforming growth factor-beta play counterregulatory roles in protein kinase C-delta-dependent control of fibroblast collagen synthesis in pulmonary fibrosis. J Biol Chem 2004;279:28315–9. Schwarz MI, King TE. Interstitial lung disease. 4th Hamilton Ontario: B.C. Decker; 2003. Xu Y, Stenmark KR, Das M, Walchak SJ, Ruff LJ, Dempsey EC. Pulmonary artery smooth muscle cells from chronically hypoxic neonatal calves retain fetal-like and acquire new growth properties. Am J Physiol Lung Cell Mol Physiol 1997;273:L234–45. Li YD, Patel JM, Block ER. NO2-induced expression of specific protein kinase C isoforms and generation of phosphatidylcholine-derived diacylglycerol in cultured pulmonary artery endothelial cells. FEBS Lett 1996;389:131–5. Murray NR, Davidson LA, Chapkin RS, Clay Gustafson W, Schattenberg DG, Fields AP. Overexpression of protein kinase C betaII induces colonic hyperproliferation and increased sensitivity to colon carcinogenesis. J Cell Biol 1999;145:699–711. Igarashi M, Wakasaki H, Takahara N, Ishii H, Jiang ZY, Yamauchi T, et al. Glucose or diabetes activates p38 mitogen-activated protein kinase via different pathways. J Clin Invest 1999;103:185–95. Devries TA, Neville MC, Reyland ME. Nuclear import of PKCdelta is required for apoptosis: identification of a novel nuclear import sequence. EMBO J 2002;21:6050–60. Chou MM, Hou W, Johnson J, Graham LK, Lee MH, Chen CS, et al. Regulation of protein kinase C zeta by PI 3-kinase and PDK-1. Curr Biol 1998;8:1069–77. Dutil EM, Toker A, Newton AC. Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1). Curr Biol 1998;8:1366–75. Le Good JA, Ziegler WH, Parekh DB, Alessi DR, Cohen P, Parker PJ. Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. Science 1998;281:2042–5. Cenni V, Doppler H, Sonnenburg ED, Maraldi N, Newton AC, Toker A. Regulation of novel protein kinase C epsilon by phosphorylation. Biochem J 2002;363:537–45. Mochly-Rosen D. Localization of protein kinases by anchoring proteins: a theme in signal transduction. Science 1995;268:247–51. Chen L, Hahn H, Wu G, Chen CH, Liron T, Schechtman D, et al. Opposing cardioprotective actions and parallel hypertrophic effects of delta PKC and epsilon PKC. Proc Natl Acad Sci USA 2001;98:11114–9. Oka N, Yamamoto M, Schwencke C, Kawabe J, Ebina T, Ohno S, et al. Caveolin interaction with protein kinase C. Isoenzyme-dependent regula-

[33]

[34]

[35]

[36]

[37]

[38] [39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

557

tion of kinase activity by the caveolin scaffolding domain peptide. J Biol Chem 1997;272:33416–21. Chakraborti S, Mandal A, Das S, Chakraborti T. Role of MMP-2 in PKCdelta-mediated inhibition of Na+ dependent Ca2+ uptake in microsomes of pulmonary smooth muscle: involvement of a pertussis toxin sensitive protein. Mol Cell Biochem 2005;280:107–17. Das M, Stenmark KR, Ruff LJ, Dempsey EC. Selected isozymes of PKC contribute to augmented growth of fetal and neonatal bovine PA adventitial fibroblasts. Am J Physiol Lung Cell Mol Physiol 1997;273:L1276– 84. Gschwendt M, Dieterich S, Rennecke J, Kittstein W, Mueller HJ, Johannes FJ. Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase c isoenzymes. FEBS Lett 1996;392: 77–80. Gray MO, Karliner JS, Mochly-Rosen D. A selective epsilon-protein kinase C antagonist inhibits protection of cardiac myocytes from hypoxiainduced cell death. J Biol Chem 1997;272:30945–51. Souroujon MC, Mochly-Rosen D. Peptide modulators of protein–protein interactions in intracellular signaling. Nat Biotechnol 1998;16:919– 24. Lahn M, Sundell K, Kohler G. The role of protein kinase C-alpha in hematologic malignancies. Acta Haematol 2006;115:1–8. Bjorkoy G, Perander M, Overvatn A, Johansen T. Reversion of Ras- and phosphatidylcholine-hydrolyzing phospholipase C-mediated transformation of NIH 3T3 cells by a dominant interfering mutant of protein kinase C lambda is accompanied by the loss of constitutive nuclear mitogenactivated protein kinase/extracellular signal-regulated kinase activity. J Biol Chem 1997;272:11557–65. Choi SH, Hyman T, Blumberg PM. Differential effect of bryostatin 1 and phorbol 12-myristate 13-acetate on HOP-92 cell proliferation is mediated by down-regulation of protein kinase Cdelta. Cancer Res 2006;66:7261–9. Leitges M, Elis W, Gimborn K, Huber M. Rottlerin-independent attenuation of pervanadate-induced tyrosine phosphorylation events by protein kinase C-delta in hemopoietic cells. Lab Invest 2001;81:1087– 95. Eichholtz T, De Bont DB, De Widt J, Liskamp RM, Ploegh HL. A myristoylated pseudosubstrate peptide, a novel protein kinase C inhibitor. J Biol Chem 1993;268:1982–6. Dorn II GW, Souroujon MC, Liron T, Chen CH, Gray MO, Zhou HZ, et al. Sustained in vivo cardiac protection by a rationally designed peptide that causes epsilon protein kinase C translocation. Proc Natl Acad Sci USA 1999;96:12798–803. Short MD, Fox SM, Lam CF, Stenmark KR, Das M. Protein kinase Czeta attenuates hypoxia-induced proliferation of fibroblasts by regulating MAP kinase phosphatase-1 expression. Mol Biol Cell 2006;17:1995–2008. Ways DK, Kukoly CA, Devente J, Hooker JL, Bryant WO, Posekany KJ, et al. MCF-7 breast cancer cells transfected with protein kinase C-alpha exhibit altered expression of other protein kinase C isoforms and display a more aggressive neoplastic phenotype. J Clin Invest 1995;95:1906–15. Romanova LY, Alexandrov IA, Nordan RP, Blagosklonny MV, Mushinski JF. Cross-talk between protein kinase C-alpha (PKC-alpha) and -delta (PKC-delta): PKC-alpha elevates the PKC-delta protein level, altering its mRNA transcription and degradation. Biochemistry (Mosc) 1998;37:5558–65. Leitges M, Plomann M, Standaert ML, Bandyopadhyay G, Sajan MP, Kanoh Y, et al. Knockout of PKC alpha enhances insulin signaling through PI3K. Mol Endocrinol 2002;16:847–58. Sun Z, Arendt CW, Ellmeier W, Schaeffer EM, Sunshine MJ, Gandhi L, et al. PKC-theta is required for TCR-induced NF-kappaB activation in mature but not immature T lymphocytes. Nature 2000;404:402–7. Simon DM, Arikan MC, Srisuma S, Bhattacharya S, Tsai LW, Ingenito EP, et al. Epithelial cell PPAR[gamma] contributes to normal lung maturation. FASEB J 2006;20:1507–9. Lahn M, Su C, Li S, Chedid M, Hanna KR, Graff JR, et al. Expression levels of protein kinase C-alpha in non-small-cell lung cancer. Clin Lung Cancer 2004;6:184–9.

558

E.C. Dempsey et al. / Pharmacological Research 55 (2007) 545–559

[51] Zuo F, Kaminski N, Eugui E, Allard J, Yakhini Z, Ben-Dor A, et al. Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans. Proc Natl Acad Sci USA 2002;99:6292–7. [52] Harvey BG, Heguy A, Leopold PL, Carolan BJ, Ferris B, Crystal RG. Modification of gene expression of the small airway epithelium in response to cigarette smoking. J Mol Med 2007;85:39–53. [53] Siflinger-Birnboim A, Johnson A. Protein kinase C modulates pulmonary endothelial permeability: a paradigm for acute lung injury. Am J Physiol Lung Cell Mol Physiol 2003;284:L435–51. [54] Ware LB, Matthay MA. Clinical practice. Acute pulmonary edema. N Engl J Med 2005;353:2788–96. [55] Daffara R, Botto L, Beretta E, Conforti E, Faini A, Palestini P, et al. Endothelial cells as early sensors of pulmonary interstitial edema. J Appl Physiol 2004;97:1575–83. [56] Harrington EO, Brunelle JL, Shannon CJ, Kim ES, Mennella K, Rounds S. Role of protein kinase C isoforms in rat epididymal microvascular endothelial barrier function. Am J Respir Cell Mol Biol 2003;28:626–36. [57] Dada LA, Chandel NS, Ridge KM, Pedemonte C, Bertorello AM, Sznajder JI. Hypoxia-induced endocytosis of Na, K-ATPase in alveolar epithelial cells is mediated by mitochondrial reactive oxygen species and PKC-zeta. J Clin Invest 2003;111:1057–64. [58] Sakao S, Taraseviciene-Stewart L, Lee JD, Wood K, Cool CD, Voelkel NF. Initial apoptosis is followed by increased proliferation of apoptosisresistant endothelial cells. FASEB J 2005;19:1178–80. [59] Littler CM, Wehling CA, Wick MJ, Fagan KA, Cool CD, Messing RO, et al. Divergent contractile and structural responses of the murine PKCepsilon null pulmonary circulation to chronic hypoxia. Am J Physiol Lung Cell Mol Physiol 2005;289:L1083–93. [60] Page K, Li J, Zhou L, Iasvovskaia S, Corbit KC, Soh JW, et al. Regulation of airway epithelial cell NF-kappa B-dependent gene expression by protein kinase C delta. J Immunol 2003;170:5681–9. [61] Rovati GE, Baroffio M, Citro S, Brichetto L, Ravasi S, Milanese M, et al. Cysteinyl-leukotrienes in the regulation of beta2-adrenoceptor function: an in vitro model of asthma. Respir Res 2006;7:103. [62] Leitges M, Gimborn K, Elis W, Kalesnikoff J, Hughes MR, Krystal G, et al. Protein kinase C-delta is a negative regulator of antigen-induced mast cell degranulation. Mol Cell Biol 2002;22:3970–80. [63] Lessmann E, Leitges M, Huber M. A redundant role for PKC-epsilon in mast cell signaling and effector function. Int Immunol 2006;18:767–73. [64] Cho SH, You HJ, Woo CH, Yoo YJ, Kim JH. Rac and protein kinase C-delta regulate ERKs and cytosolic phospholipase A2 in FcepsilonRI signaling to cysteinyl leukotriene synthesis in mast cells. J Immunol 2004;173:624–31. [65] Hewson CA, Edbrooke MR, Johnston SL. PMA induces the MUC5AC respiratory mucin in human bronchial epithelial cells, via PKC, EGF/TGF-alpha, Ras/Raf, MEK, ERK and Sp1-dependent mechanisms. J Mol Biol 2004;344:683–95. [66] Zhou L, Hershenson MB. Mitogenic signaling pathways in airway smooth muscle. Respir Physiol Neurobiol 2003;137:295–308. [67] Pang L, Nie M, Corbett L, Donnelly R, Gray S, Knox AJ. Protein kinase C-epsilon mediates bradykinin-induced cyclooxygenase-2 expression in human airway smooth muscle cells. FASEB J 2002;16:1435–7. [68] Zhang HP, Xu YJ, Zhang ZX, Ni W, Chen SX. Expression of protein kinase C and nuclear factor kappa B in lung tissue of patients with chronic obstructive pulmonary disease. [Zhonghua nei ke za zhi] Chin J Intern Med 2004;43:756–9. [69] Zhang J, Anastasiadis PZ, Liu Y, Thompson EA, Fields AP. Protein kinase C (PKC) betaII induces cell invasion through a Ras/Mek-, PKC iota/Rac 1-dependent signaling pathway. J Biol Chem 2004;279:22118–23. [70] Carter CA. Protein kinase C as a drug target: implications for drug or diet prevention and treatment of cancer. Curr Drug Targets 2000;1:163– 83. [71] Kim CF, Jackson EL, Woolfenden AE, Lawrence S, Babar I, Vogel S, et al. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 2005;121:823–35. [72] Watkins DN, Berman DM, Burkholder SG, Wang B, Beachy PA, Baylin SB. Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer. Nature 2003;422:313–7.

[73] Catley MC, Cambridge LM, Nasuhara Y, Ito K, Chivers JE, Beaton A, et al. Inhibitors of protein kinase C (PKC) prevent activated transcription: role of events downstream of NF-kappaB DNA binding. J Biol Chem 2004;279:18457–66. [74] Tortora G, Ciardiello F. Antisense strategies targeting protein kinase C: preclinical and clinical development. Semin Oncol 2003;30:26–31. [75] Wang XY, Repasky E, Liu HT. Antisense inhibition of protein kinase Calpha reverses the transformed phenotype in human lung carcinoma cells. Exp Cell Res 1999;250:253–63. [76] Carducci MA, Musib L, Kies MS, Pili R, Truong M, Brahmer JR, et al. Phase I dose escalation and pharmacokinetic study of enzastaurin, an oral protein kinase C beta inhibitor, in patients with advanced cancer. J Clin Oncol 2006;24:4092–9. [77] Clark AS, West KA, Blumberg PM, Dennis PA. Altered protein kinase C (PKC) isoforms in non-small cell lung cancer cells: PKCdelta promotes cellular survival and chemotherapeutic resistance. Cancer Res 2003;63:780–6. [78] Ding L, Wang H, Lang W, Xiao L. Protein kinase C-epsilon promotes survival of lung cancer cells by suppressing apoptosis through dysregulation of the mitochondrial caspase pathway. J Biol Chem 2002;277:35305–13. [79] Sonnemann J, Gekeler V, Ahlbrecht K, Brischwein K, Liu C, Bader P, et al. Down-regulation of protein kinase Ceta by antisense oligonucleotides sensitises A549 lung cancer cells to vincristine and paclitaxel. Cancer Lett 2004;209:177–85. [80] Regala RP, Weems C, Jamieson L, Khoor A, Edell ES, Lohse CM, et al. Atypical protein kinase C iota is an oncogene in human non-small cell lung cancer. Cancer Res 2005;65:8905–11. [81] Lai CK, Wallace WD, Fishbein MC. Histopathology of pulmonary fibrotic disorders. Semin Respir Crit Care Med 2006;27:613–22. [82] Khalil N, Bereznay O, Sporn M, Greenberg AH. Macrophage production of transforming growth factor beta and fibroblast collagen synthesis in chronic pulmonary inflammation. J Exp Med 1989;170:727–37. [83] Broekelmann TJ, Limper AH, Colby TV, Mcdonald JA. Transforming growth factor beta 1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis. Proc Natl Acad Sci USA 1991;88:6642–6. [84] Wu T, Han C, Shelhamer JH. Involvement of p38 and p42/44 MAP kinases and protein kinase C in the interferon-gamma and interleukin1alpha-induced phosphorylation of 85-kDa cytosolic phospholipase A(2) in primary human bronchial epithelial cells. Cytokine 2004;25:11–20. [85] Wyatt TA, Heires AJ, Sanderson SD, Floreani AA. Protein kinase C activation is required for cigarette smoke-enhanced C5a-mediated release of interleukin-8 in human bronchial epithelial cells. Am J Respir Cell Mol Biol 1999;21:283–8. [86] Kontny E, Ziolkowska M, Ryzewska A, Maslinski W. Protein kinase c-dependent pathway is critical for the production of pro-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6). Cytokine 1999;11:839–48. [87] Kohyama T, Wyatt TA, Liu X, Wen FQ, Kobayashi T, Fang Q, et al. PGD(2) modulates fibroblast-mediated native collagen gel contraction. Am J Respir Cell Mol Biol 2002;27:375–81. [88] Marsland BJ, Nembrini C, Schmitz N, Abel B, Krautwald S, Bachmann MF, et al. Innate signals compensate for the absence of PKC-{theta} during in vivo CD8(+) T cell effector and memory responses. Proc Natl Acad Sci USA 2005;102:14374–9. [89] Pitt BR, Ortiz LA. Stem cells in lung biology. Am J Physiol Lung Cell Mol Physiol 2004;286:L621–3. [90] Davie NJ, Crossno Jr JT, Frid MG, Hofmeister SE, Reeves JT, Hyde DM, et al. Hypoxia-induced pulmonary artery adventitial remodeling and neovascularization: contribution of progenitor cells. Am J Physiol Lung Cell Mol Physiol 2004;286:L668–78. [91] Ishizawa K, Kubo H, Yamada M, Kobayashi S, Numasaki M, Ueda S, et al. Bone marrow-derived cells contribute to lung regeneration after elastase-induced pulmonary emphysema. FEBS Lett 2004;556:249– 52. [92] Yamada M, Kubo H, Ishizawa K, Kobayashi S, Shinkawa M, Sasaki H. Increased circulating endothelial progenitor cells in patients with bacterial pneumonia: evidence that bone marrow derived cells contribute to lung repair. Thorax 2005;60:410–3.

E.C. Dempsey et al. / Pharmacological Research 55 (2007) 545–559 [93] Doerschuk CM. Circulating endothelial progenitor cells in pulmonary inflammation. Thorax 2005;60:362–4. [94] Schmidt EK, Fichelson S, Feller SM. PI3 kinase is important for Ras, MEK and Erk activation of Epo-stimulated human erythroid progenitors. BMC Biol 2004;2:7. [95] Koyanagi M, Haendeler J, Badorff C, Brandes RP, Hoffmann J, Pandur P, et al. Non-canonical Wnt signaling enhances differentiation of human circulating progenitor cells to cardiomyogenic cells. J Biol Chem 2005;280:16838–42. [96] Hales CA, Kradin RL, Brandstetter RD, Zhu YJ. Impairment of hypoxic pulmonary artery remodeling by heparin in mice. Am Rev Respir Dis 1983;128:747–51. [97] Ahmed T, Gonzalez BJ, Danta I. Prevention of exercise-induced bronchoconstriction by inhaled low-molecular-weight heparin. Am J Respir Crit Care Med 1999;160:576–81. [98] Gunther A, Lubke N, Ermert M, Schermuly RT, Weissmann N, Breithecker A, et al. Prevention of bleomycin-induced lung fibrosis by aerosolization of heparin or urokinase in rabbits. Am J Respir Crit Care Med 2003;168:1358–65. [99] Barzu T, Herbert JM, Desmouliere A, Carayon P, Pascal M. Characterization of rat aortic smooth muscle cells resistant to the antiproliferative activity of heparin following long-term heparin treatment. J Cell Physiol 1994;160:239–48. [100] Herbert JM, Clowes M, Lea HJ, Pascal M, Clowes AW. Protein kinase C alpha expression is required for heparin inhibition of rat smooth muscle cell proliferation in vitro and in vivo. J Biol Chem 1996;271:25928–35. [101] Pukac LA, Ottlinger ME, Karnovsky MJ. Heparin suppresses specific second messenger pathways for protooncogene expression in rat vascular smooth muscle cells. J Biol Chem 1992;267:3707–11. [102] Das M, Stenmark KR, Dempsey EC. Enhanced growth of fetal and neonatal pulmonary artery adventitial fibroblasts is dependent on protein kinase C. Am J Physiol Lung Cell Mol Physiol 1995;269:L660–7.

559

[103] Yan W, Tiruppathi C, Lum H, Qiao R, Malik AB. Protein kinase C beta regulates heterologous desensitization of thrombin receptor (PAR-1) in endothelial cells. Am J Physiol Cell Physiol 1998;274:C387–95. [104] Nixon JS, Bishop J, Bradshaw D, Davis PD, Hill CH, Elliott LH, et al. Novel, potent and selective inhibitors of protein kinase C show oral anti-inflammatory activity. Drugs Exp Clin Res 1991;17:389–93. [105] Salonen T, Sareila O, Jalonen U, Kankaanranta H, Tuominen R, Moilanen E. Inhibition of classical PKC isoenzymes downregulates STAT1 activation and iNOS expression in LPS-treated murine J774 macrophages. Br J Pharmacol 2006;147:790–9. [106] Smith CP, Oh JD, Bibbiani F, Collins MA, Avila I, Chase TN. Tamoxifen effect on L-DOPA induced response complications in parkinsonian rats and primates. Neuropharmacology 2007;52:515–26. [107] Sun MK, Alkon DL. Bryostatin-1: pharmacology and therapeutic potential as a CNS drug. CNS Drug Rev 2006;12:1–8. [108] Goekjian PG, Jirousek MR. Protein kinase C inhibitors as novel anticancer drugs. Expert Opin Investig Drugs 2001;10:2117–40. [109] Mckay RA, Miraglia LJ, Cummins LL, Owens SR, Sasmor H, Dean NM. Characterization of a potent and specific class of antisense oligonucleotide inhibitor of human protein kinase C-alpha expression. J Biol Chem 1999;274:1715–22. [110] Khasar SG, Lin YH, Martin A, Dadgar J, Mcmahon T, Wang D, et al. A novel nociceptor signaling pathway revealed in protein kinase C epsilon mutant mice. Neuron 1999;24:253–60. [111] Huang F, Subbaiah PV, Holian O, Zhang J, Johnson A, Gertzberg N, et al. Lysophosphatidylcholine increases endothelial permeability: role of PKCalpha and RhoA cross talk. Am J Physiol Lung Cell Mol Physiol 2005;289:L176–85. [112] Brehmer D, Godl K, Zech B, Wissing J, Daub H. Proteome-wide identification of cellular targets affected by bisindolylmaleimide-type protein kinase C inhibitors. Mol Cell Proteomics 2004;3:490–500.