Effect of ageing on the expression of protein kinase C and its activation by 1,25(OH)2-vitamin D3 in rat skeletal muscle

To characterize age-induced effects on muscle protein kinase C (PKC) and its regulation by the steroid hormone 1,25(OH)2-vitamin D3 [1,25(OH)2D3], changes in PKC activity and the expression and translocation of the specific PKC conventional isoforms alpha and beta, novel isoforms delta, epsilon, and theta and atypical isoform zeta were studied in homogenates and subcellular fractions from skeletal muscle of young (3 months) and aged (24 months) rats treated in vitro with 1,25(OH)2D3. The hormone (10(-9) M) increased total and membrane PKC activity, within 1 min, and these effects were completely blunted in muscle from aged rats. The presence of PKC isoenzymes was shown by Western blot analysis with the use of specific antibodies. The expression of PKC alpha, beta and delta was greatly diminished in old rats, whereas age-related changes were less pronounced in the isoforms epsilon, theta and zeta. After a short exposure (1 min) of muscle to 1,25(OH)2D3, increased amounts of PKC alpha and beta in muscle membranes and reverse translocation (from membrane to cytosol) of PKC epsilon were observed only in young animals. The data indicate that, in rat muscle, ageing impairs calcium-dependent PKC (alpha and beta) and calcium-independent PKC (delta, epsilon, theta and zeta) signal transduction pathways under selective regulation by 1,25(OH)2D3.     

Differential action of nerve growth factor and phorbol ester TPA on rat synaptosomal PKC isoenzymes.

The subcellular redistribution of protein kinase C family members (alpha, beta, gamma, delta, epsilon and zeta isoforms) was examined in response to treatment with 12-O-tetradecanoyl-phorbol-13 acetate (TPA) or nerve growth factor (NGF) in a synaptosomal-enriched P2 fraction from rat brain. Treatment with TPA affected members of the classical-PKC family (alpha, beta and gamma), resulting in a final loss of total protein of each isoenzyme. The kinetics of changes of members of the novel-PKC family are different, the delta isoform being translocated, but not down-regulated, while the epsilon isoform showing only a slight diminishing of immunoreactivity in the soluble and particulate fractions. The atypical-PKC zeta isoform was not translocated in response to TPA. Incubation with NGF induced a loss of immunoreactivity of the cytosolic alpha, beta and epsilon isoforms, but the membrane fractions of these isoforms were not appreciably affected. In contrast, a marked translocation from cytosol to membrane was observed in the case of the gamma and delta isoforms. The zeta isoform presented a slight translocation from the particulate fraction to the soluble fraction. Thus, the results show that the effects of TPA and NGF on PKC isoforms are not coincident in synaptosomes, the 6 isoform being activated and not down-regulated by both treatments, whereas the gamma isoform is only down-regulated in the case of TPA, but presents sustained translocation with NGF, indicating that PKC isoform-specific degradation pathways exist in synaptic terminals. The effects of NGF on PKC isoforms coexist with an increase in NGF-induced polyphosphoinositide hydrolysis, suggesting the participation of phospholipases.     

Multiple isoforms of protein kinase C in lymphocytes and airway smooth muscle of guinea pig

The isoenzyme pattern of protein kinase C (PKC) in lymphocytes and airway smooth muscles (ASM) was examined by Western blot using commercially available monoclonal antibodies. The results showed the presence of PKC alpha, beta, gamma, epsilon, eta, mu and zeta in lymphocytes and PKC alpha, gamma, epsilon, eta and zeta in ASM. The unexpected feature was the presence of PKCgamma in both lymphocytes and ASM of guinea pigs. Expression of this PKC isoform is usually restricted to tissues in the central nervous system or spinal cord. Expression of PKC delta, theta, lambda and tau was not detected in either lymphocytes or ASM.     

Expression of four protein kinase C isoforms in rat fibroblasts. Distinct subcellular distribution and regulation by calcium and phorbol esters.

Protein kinase C (PKC), the major receptor for tumor-promoting phorbol esters, consists of a family of at least eight distinct lipid-regulated enzymes. How the various PKC isozymes are regulated in vivo and how they couple to particular cellular responses is largely unknown. We have examined the expression and regulation of PKC isoforms in R6 rat embryo fibroblasts. Northern and Western blot analyses indicate that these cells express four PKC isoforms, cPKC alpha, nPKC epsilon, nPKC delta, and nPKC zeta; of which nPKC epsilon and nPKC delta are the most abundant. In agreement with the simultaneous presence of cPKC and nPKC isozymes, both Ca(2+)-dependent and -independent PKC activities were detected in extracts of these cells. cPKC alpha and nPKC zeta were predominantly localized in the cytosol when subcellular fractionation was carried out in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. When cell lysis was carried out in the presence of Ca2+, greater than 50% of cPKC alpha redistributed to the particulate fraction, whereas nPKC zeta remained in the cytosol. In contrast to cPKC alpha and nPKC zeta, 60-80% of nPKC epsilon and nPKC delta were located in a Ca(2+)-insensitive, membrane-bound form. Treatment of R6 cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA), resulted in the translocation of all four PKC isozymes to the membrane fraction, and the subsequent down-regulation of cPKC alpha, nPKC zeta, and nPKC delta, nPKC epsilon, however, was only partially down-regulated in response to long-term TPA exposure. Overproduction of exogenous cPKC beta I in R6 cells conferred partial resistance of nPKC delta to TPA-induced down-regulation and potentiated the resistance of nPKC epsilon to down-regulation. These results demonstrate that the multiple isoforms of PKC which coexist within a single cell type are differentially regulated by extra- and intracellular stimuli and may thereby influence growth control and transformation via distinct mechanisms.     

Prevention of NMDA-induced death of cortical neurons by inhibition of protein kinase Czeta

Excitotoxicity through stimulation of N-methyl-d-aspartate (NMDA) receptors contributes to neuronal death in brain injuries, including stroke. Several lines of evidence suggest a role for protein kinase C (PKC) isoforms in NMDA excitotoxicity. We have used specific peptide inhibitors of classical PKCs (alpha, beta, and gamma), novel PKCs delta and epsilon, and an atypical PKCzeta in order to delineate which subspecies are involved in NMDA-induced cell death. Neuronal cell cultures were prepared from 15-day-old mouse embryos and plated onto the astrocytic monolayer. After 2 weeks in vitro the neurons were exposed to 100 micro m NMDA for 5 min, and 24 h later the cell viability was examined by measuring the lactate dehydrogenase release and bis-benzimide staining. While inhibitors directed to classical (alpha, beta, and gamma) or novel PKCs (delta or epsilon) had no effect, the PKCzeta inhibitor completely prevented the NMDA-induced necrotic neuronal death. Confocal microscopy confirmed that NMDA induced PKCzeta translocation, which was blocked by the PKCzeta inhibitor. The NMDA-induced changes in intracellular free Ca2+ were not affected by the peptides. In situ hybridization experiments demonstrated that PKCzeta mRNA is induced in the cortex after focal brain ischemia. Altogether, the results indicate that PKCzeta activation is a downstream signal in NMDA-induced death of cortical neurons.     

Early and delayed glutamate effects in rat primary cortical neurons. Changes in the subcellular distribution of protein kinase C isoforms and in intracellular calcium concentration

Glutamate-induced changes in the subcellular distribution of protein kinase C isoforms and in the intracellular calcium concentration were investigated in rat primary cortical neurons. Western blot analysis of protein kinase C isoforms (alpha, beta1, beta2, gamma, delta, epsilon, zeta and theta), performed 30 min after a 10 min treatment with 30 microM glutamate, revealed a decrease in the total beta1 (-24%) and beta2 (-40%) isoform levels, without any significant change in any of the other isozymes. All conventional isoforms translocated to the membrane compartment, while delta, epsilon, zeta and theta; maintained their initial subcellular distribution. Twenty-four hours after glutamate treatment, the total protein kinase C labelling had increased, particularly the epsilon isoform, which accounted for 34% of the total densitometric signal. At this time, protein kinase C beta1, delta, epsilon and zeta isoforms were mainly detected in the membrane compartment, while gamma and theta; signals were displayed almost solely in the cytosol. Basal intracellular calcium concentration (FURA 2 assay) was concentration-dependently increased (maximum effect +77%) 30 min, but not 24h after a 10 min glutamate (10-100 microM) treatment, while the net increase induced by electrical stimulation (10 Hz, 10s) was consistently reduced (maximum effect -64%). The N-methyl-d-aspartate receptor antagonist, MK-801, 1 microM, prevented glutamate action both 30 min and 24 h after treatment, while non-selective protein kinase C inhibitors, ineffective at 30 min, potentiated it at 24 h. These findings show that protein kinase C isoforms are differently activated and involved in the early and delayed glutamate actions, and that the prevailing effect of their activation is neuroprotective.     

Redistribution of protein kinase C isoforms in rat pancreatic acini during lactation and weaning

Freshly enzymatically isolated pancreatic acini from lactating and weaning Wistar rats were used to investigate the role of protein kinase C (PKC) isoforms during these physiologically relevant pancreatic secretory and growth processes. The combination of immunoblot and immunohistochemical analysis shows that the PKC isoforms alpha, delta, and epsilon are present in pancreatic acini from control, lactating and weaning rats. A vesicular distribution of PKC-alpha, -delta, and -epsilon was detected by immunohistochemical analysis in the pancreatic acini from all the experimental groups. PKC-delta showed the strongest PKC immunoreactivity (PKC-IR). In this vesicular distribution, PKC-IR was located at the apical region of the acinar cells. No differences were observed between control, lactating and weaning rats. However, the immunoblot analysis of pancreatic PKC isoforms during lactation and weaning showed a significant translocation of PKC-delta from the cytosol to the membrane fraction when compared with control animals. Translocation of PKC isoforms (alpha, delta and epsilon) in response to 12-O-tetradecanoyl phorbol 13-acetate (TPA) 1 microM (15 min, 37 degrees C) was comparable in pancreatic acini from control, lactating and weaning rats. In the control group, a significant translocation of all the isoforms (alpha, delta and epsilon) from the cytosol to the membrane was observed. The PKC isoform most translocated by TPA was PKC-delta. In contrast, no statistically significant increase in PKC-delta translocation was detected in pancreatic acini isolated from lactating or weaning rats. These results suggest that the PKC isoforms are already translocated to the surface of the acinar cells from lactating or weaning rats. In addition, they suggest that isoform specific spatial PKC distribution and translocation occur in association with the growth response previously described in the rat exocrine pancreas during lactation and weaning.     

Fatty acid regulation of protein kinase C isoforms in prostate cancer cells

Polyunsaturated fatty acids influence the aetiology of prostate cancer. Their effects on cellular mechanisms regulating prostate tumorigenesis are unclear. Using prostate cancer cells (LNCaP), we determined effects of n-9-OA, n-6-LA, and n-3-EPA on total PKC and its isoforms in relation to cell proliferation and PSA production. PKC-alpha, delta, gamma, iota, mu, and zeta were present in LNCaP cells; PKC-beta, epsilon, eta, and theta isoforms were not. PKC-alpha was detected only in cytosol; PKC-delta, iota, gamma, and mu were present in cytosol and in membranes. Fatty acids increased cell proliferation, total PKC activity and elicited pro-proliferative effects on specific PKC isoforms (PKC-delta and -iota). EPA and LA increased total PKC activity and reduced membrane-abundance of PKC-delta. OA reduced cytosolic and membrane PKC-delta. Only EPA reduced PKC-gamma membrane abundance. Fatty acids enhanced cytosolic PKC-iota abundance but only EPA and to a lesser extent LA increased its membrane content. Changes in PKC-delta, -iota, and -gamma did not affect PSA production.     

Expression and localization of protein kinase C theta isoform in mouse testis.

Protein kinase C (PKC) is encoded by a complex of a gene family, and its multiple isoforms are expressed in various mammalian tissues. The objective of this study was to investigate the expression and localization of a PKC theta isoform in mouse testis. PKC theta displays the highest homology to PKC delta, lacks the Ca2+-binding C2 domain and, thus, belongs to the subfamily of Ca2+-independent PKC enzymes which also includes the delta, epsilon, zeta and eta isoforms. We analyzed the PKC theta mRNA and protein by Northern blotting, in situ hybridization, and immunohistochemistry. In testes of normal mice, signals of PKC theta isoform expression were detected specifically in the interstitial cells of testes. The expression of PKC theta isoform was also detected in testes of germ cell-deficient W/W(v) mice. These results suggest that PKC theta isoform has the specific biological functions in the interstitial cells of testis.     

Differential requirement for classic and novel PKC isoforms in respiratory burst and phagocytosis in RAW 264.7 cells

The binding of Ab (IgG)-opsonized particles by FcgammaRs on macrophages results in phagocytosis of the particles and generation of a respiratory burst. Both IgG-stimulated phagocytosis and respiratory burst involve activation of protein kinase C (PKC). However, the specific PKC isoforms required for these responses have yet to be identified. We have studied the involvement of PKC isoforms in IgG-mediated phagocytosis and respiratory burst in the mouse macrophage-like cell line, RAW 264.7. Like primary monocyte/macrophages, their IgG-mediated phagocytosis was calcium independent and diacylglycerol sensitive, consistent with novel PKC activation. Respiratory burst in these cells was Ca2+ dependent and inhibited by staurosporine and calphostin C as well as by the classic PKC-selective inhibitors G? 6976 and CGP 41251, suggesting that classic PKC is required. In contrast, phagocytosis was blocked by general PKC inhibitors but not by the classic PKC-specific drugs. RAW 264.7 cells expressed PKCs alpha, betaI, delta, epsilon, and zeta. Subcellular fractionation demonstrated that PKCs alpha, delta, and epsilon translocate to membranes during phagocytosis. In Ca2+-depleted cells, only novel PKCs delta and epsilon increased in membranes, and the time course of their translocation was consistent with phagosome formation. Confocal microscopy of cells transfected with green fluorescent protein-conjugated PKC alpha or epsilon confirmed that these isoforms translocated to the forming phagosome in Ca-replete cells, but only PKC epsilon colocalized with phagosomes in Ca2+-depleted cells. Taken together, these results suggest that the classic PKC alpha mediates IgG-stimulated respiratory burst in macrophages, whereas the novel PKCs delta and/or epsilon are necessary for phagocytosis.     

The epsilon isoform of protein kinase C is involved in regulation of the LTD(4)-induced calcium signal in human intestinal epithelial cells

We investigated the potential roles of specific isoforms of protein kinase C (PKC) in the regulation of leukotriene D(4)-induced Ca(2+) signaling in the intestinal epithelial cell line Int 407. RT-PCR and Western blot analysis revealed that these cells express the PKC isoforms alpha, betaII, delta, epsilon, zeta, and mu, but not betaI, gamma, eta, or theta;. The inflammatory mediator leukotriene D(4) (LTD(4)) caused the TPA-sensitive PKC isoforms alpha, delta, and epsilon, but not betaII, to rapidly translocate to a membrane-enriched fraction. The PKC inhibitor GF109203X at 30 microM but not 2 microM significantly impaired the LTD(4)-induced Ca(2+) signal, indicating that the response involves a novel PKC isoform, such as delta or epsilon, but not alpha. LTD(4)-induced Ca(2+) signaling was significantly suppressed in cells pretreated with TPA for 15 min and was abolished when the pretreatment was prolonged to 2 h. Immunoblot analysis revealed that the reduction in the LTD(4)-induced calcium signal coincided with a reduction in the cellular content of PKCepsilon and, to a limited extent, PKCdelta. LTD(4)-induced Ca(2+) signaling was also markedly suppressed by microinjection of antibodies against PKCepsilon but not PKCdelta. These data suggest that PKCepsilon plays a unique role in regulation of the LTD(4)-dependent Ca(2+) signal in intestinal epithelial cells.     

Analysis of protein kinase C isoforms involved in the activation of laminin receptor in Raw264.7 macrophages

Adherence of hematopoietic macrophages to a laminin (LM) substratum requires protein kinase C (PKC)-dependent activation of LM receptor. This study was performed to analyze PKC isoform(s) leading to the activation of LM receptor during Raw264.7 macrophage-like cell adhesion to a LM substratum. Raw264.7 cells expressed multiple PKC isoforms, including alpha, beta I, delta, epsilon, zeta, lambda/iota, and mu. Among the PKC isoforms expressed, selective activation of PKC delta and epsilon was sufficient to induce cell adhesion to LM. PKC-dependent cell adherence was blocked by the selective inhibition of PKC delta, suggesting that PKC delta was the responsible PKC isoform leading to activation of LM receptor. PKC delta appeared to activate LM receptor in an intact microfilament-dependent pathway, because disruption of microfilament inhibited cell adhesion to LM without affecting PKC delta activation.     

Evidence for clonal heterogeneity of the expression of six protein kinase C isoforms in murine B and T lymphocytes.

Protein kinase C (PKC), which plays a pivotal role in lymphocyte activation, represents a homologous family of at least nine proteins. Seven genes that encode PKC proteins have been identified. Since the regulatory properties and substrate specificities of the isoforms are not identical in vitro, it is possible that each isoform plays a unique role in cell activation. Toward an understanding of the role of PKC isoforms in lymphocyte activation we have studied the expression of mRNA encoding six of the isoforms (alpha, beta, gamma, delta, epsilon, and zeta) in T cell clones and B cell lines. PKC isoform phenotyping was done by MAPPing using isoform-specific primers and slot-blot analyses of mRNA were performed using specific probes. T cell clones and B cell lines were determined to express levels of the delta, epsilon, and zeta isoforms of PKC that were detectable by MAPPing. Plasmacytomas did not express PKC-beta message detectable by MAPPing. Slot blot analyses and Western blot analyses with peptide-specific antibody confirmed that B cell plasmacytomas did not express PKC-beta mRNA or protein. T cell clones and B cell lines were similar in that none expressed PKC-gamma. In cells that expressed PKC isoforms that were detectable by the MAPPing protocol, there was heterogeneity in the relative abundance of isoform mRNA (PKC-delta and -beta) and protein (PKC-beta and -epsilon). Such diversity of isoform expression could be responsible for the differential responsiveness of lymphocyte clones to activating stimuli.     

Differential changes in protein kinase C associated with regeneration of rat extensor digitorum longus and soleus muscles

We used a model of crush-induced regeneration in rat in order to characterize biochemically and histologically the implication of protein kinase C (PKC) in muscle repair after damage. In this model, slow soleus and fast extensor digitorum longus (EDL) muscle regeneration proceed differently. PKC activity has been assayed in regenerating muscles and their intact contralateral during the first 14 days following crushing. Degeneration (myolysis) occurring shortly after crush was associated with a marked down-regulation of the enzyme in both wound muscles and notable increase in the corresponding contralateral muscles. Muscle fiber reconstruction in EDL was associated with a rise in PKC activity which peaked at day 7 in regenerating muscle where it was twice higher than in intact muscle. At variance, muscle PKC activity in soleus increased slower than that of EDL and reached later intact level. Western blot analysis and immunohistochemical studies of representative members of the three PKC subfamilies were performed. All the isoform tested were much less expressed in regenerating than in control intact muscles suggesting that the overall PKC activity in regenerating muscles was more activable than in controls. We have shown that PKC isoforms were sequentially expressed during regeneration in both muscle types. PKC theta; being present the earliest, then delta, epsilon and alpha and finally zeta, beta and eta. Some isoforms were differentially expressed according muscle type. PKC delta being more expressed in soleus whereas beta and eta appeared earlier in EDL. Histochemical studies have revealed that the isoforms were differently localized in muscle tissue and that fiber regeneration was associated with PKC alpha translocation from sarcoplasma to sarcolemma. Together these data have shown that multiple PKC isoforms are implicated in the regenerative process acting at different in times and location and suggesting that individual isoform may fulfill distinct functions.     

The epsilon-isoform of PKC mediates the hypertonic activation of cation channels in confluent monolayers of rat hepatocytes.

We were interested whether PKC alpha, delta, epsilon or zeta is the isoform actually employed in the activation of hypertonicity-induced cation channels (HICCs) in primary cultures of rat hepatocytes. Quantitative SDS-page and Western-blot experiments revealed that PKC alpha, delta and epsilon were stimulated by Indolactam V (as a DAG substitute for activation of c and nPKCs) but that only PKC delta and epsilon did respond to hypertonic stress. Furthermore, chelation of intracellular Ca(++) by BAPTA-AM did not alter HICC activation in cable-analysis experiments whereas Indolactam V as well as V8 (an Indolactam derivative specific for PKC delta and epsilon) activated HICC currents under isotonic conditions. Finally, by use of Rottlerin (as an inhibitor exhibiting a slight preference for PKC delta over epsilon) PKC epsilon could be identified as the most likely isoform responsible for the activation of the HICC.     

Respiratory syncytial virus infection results in activation of multiple protein kinase C isoforms leading to activation of mitogen-activated protein kinase

Respiratory syncytial virus (RSV) is an important respiratory pathogen that preferentially infects epithelial cells in the airway and causes a local inflammatory response. Very little is known about the second messenger pathways involved in this response. To characterize some of the acute response pathways involved in RSV infection, we used cultured human epithelial cells (A549) and optimal tissue culture-infective doses (TCID(50)) of RSV. We have previously shown that RSV-induced IL-8 release is linked to activation of the extracellular signal-related kinase (ERK) mitogen-activated protein kinase pathway. In this study, we evaluated the upstream events involved in ERK activation by RSV. RSV activated ERK at two time points, an early time point consistent with viral binding and a later sustained activation consistent with viral replication. We next evaluated the role of protein kinase C (PKC) isoforms in RSV-induced ERK kinase activity. We found that A549 cells contain the Ca(2+)-dependent isoforms alpha and beta1, and the Ca(2+)-independent isoforms delta, epsilon, eta, mu, theta, and zeta. Western analysis showed that RSV caused no change in the amounts of these isoforms. However, kinase activity assays demonstrated activation of isoform zeta within 10 min of infection, followed by a sustained activation of isoforms beta1, delta, epsilon, and mu 24-48 h postinfection. A cell-permeable peptide inhibitor specific for the zeta isoform decreased early ERK kinase activation by RSV. Down-regulation of the other PKC isoforms with PMA blocked the late sustained activation of ERK by RSV. These studies suggest that RSV activates multiple PKC isoforms with subsequent downstream activation of ERK kinase.     

Associations of PKC isoforms with the cytoskeleton of B16F10 melanoma cells.

Although PKC plays a major role in regulating the morphology and function of the cytoskeleton, little is known about in situ associations of specific isoforms with the cytoskeleton. We demonstrate that seven PKC isoforms are expressed in B16F10 melanoma cells and show different levels of induction by serum. Using cell cytoskeleton preparations (CSKs), confocal microscopy, and immunocytochemistry, all isoforms show specific patterns of localization to focal contact-like structures (alpha, delta), very small cytoplasmic granules/vesicles (all isoforms), dense ordered arrays of small granules in the perinuclear region (alpha, delta), granules/vesicles associated with a homogeneous framework in the cytoplasm adjacent to the nucleus (gamma), or irregular-shaped patches of granules at or near the nuclear perimeter (eta, theta). In addition, several isoforms are present as cytoplasmic granules/ vesicles in linear or curvilinear arrays (alpha, delta, epsilon, theta). When isoform localization is examined using 3.7% formaldehyde or methanol:acetone, the patterns of localization in CSKs are often difficult or impossible to detect, and many are described here for the first time. Double-labeling experiments with CSK demonstrate that PKC actin co-localizes with punctate alpha-rich particles above the nucleus, granules of epsilon throughout the cytoplasm, and with theta in irregular-shaped aggregates associated with the nucleus. Vimentin co-localizes with perinuclear granules of delta and beta(2), and alpha-tubulin co-localizes with theta in structures at or near the nuclear surface and in microtubules associated with the microtubule organizing center (MTOC). In summary, the present study demonstrates that seven PKC isoforms are endogenously expressed in B16F10 melanoma cells. These isoforms show various levels of induction by serum and specific patterns of association with various components of the detergent-resistant cell cytoskeleton.     

Alterations in the expression and localization of protein kinase C isoforms during mammary gland differentiation.

Protein kinase C (PKC) is involved in signaling that modulates the proliferation and differentiation of many cell types, including mammary epithelial cells. In addition, changes in PKC expression or activity have been observed during mammary carcinogenesis. In order to examine the involvement of specific PKC isoforms during normal mammary gland development, the expression and localization of PKCs alpha, delta, epsilon and zeta were examined during puberty, pregnancy, lactation, and involution. By immunoblot analysis, expression of PKC alpha, delta, epsilon and zeta proteins was increased in mammary epithelial organoids during the transition from puberty to pregnancy. In mammary gland frozen sections, PKCs alpha, delta, epsilon and zeta were stained in the luminal epithelium and myoepithelium, in varying isoform-and developmental stage-specific locations. PKC alpha was found in a punctate apical localization in the luminal epithelium during pregnancy. During lactation, PKC epsilon was present in the nucleus, and PKC zeta was concentrated in the subapical region of the luminal epithelium. Additionally, marked staining for PKCs alpha, delta, epsilon, and zeta was observed in the myoepithelial cells at the base of ducts and alveoli. This basal ductal and alveolar staining differed in intensity in a developmentally-specific fashion. During most time points (virgin, pregnant, lactating, and early involution), myoepithelial cells of the duct were more intensely stained than those lining the alveoli for PKCs alpha, delta, epsilon and zeta. During late involution (days 9-12), the preferential staining of ducts was lost or reversed, and the myoepithelial cells lining the regressing alveolar structures stained equally (PKCs epsilon and zeta) or more intensely (PKCs alpha and delta), coincident with the thickening of the myoepithelial cells surrounding the regressing alveoli. The increased PKC isoform staining at the base of alveoli during involution suggests that alveolar regression may be influenced by alterations in signaling in the alveolar myoepithelium.