Alterations in estrogen receptor isoforms in the mammary gland and uterus of the rat during differentiation

1. Estrogen receptors in lactating mammary glands and uteri of rats which were 10 and 19 days postpartum exhibited molecular heterogeneity based on their surface charge properties. 2. The polymorphism of estrogen receptors detected by high-performance ion exchange chromatography may be monitored in-line with a radioisotope detector. 3. Estrogen receptors from the mammary gland and uterus of rats at 10 days of lactation exhibited primarily two receptor isoforms eluting at 200-250 mM and 250-300 mM phosphate, whereas three ionic isoforms (eluting at 50-150, 200-250 and 325-375 mM phosphate) were found in the mammary glands of rats at 19 days of lactation. Similar changes in the profiles of estrogen receptor isoforms were observed in uterine cytosol preparations at each stage of postpartum differentiation. 4. The elution pattern of receptor-associated radioactivity was not altered by the addition of diisopropylphosphate, a potent inhibitor of trypsin-like proteases, either before, during or immediately after homogenization. This indicates that the differences observed in the receptor elution profile of 10 and 19 day postpartum lactating mammary glands were not due to artifactual proteolysis. 5. In summary, our data indicate that the differentiation stage of lactating mammary glands may dictate the final profile of receptor isoforms detected.     

Differential localization of conventional protein kinase C isoforms during mouse oocyte development

Protein kinase C (PKC), the major cell target for tumor-promoting phorbol esters, plays a central role in signal transduction pathways. In many biological systems where Ca(2+) serves as a second messenger, regulatory control is mediated by PKC. The activation of PKC depends on its binding to RACK1 receptor, which is an intracellular protein anchor for activated PKC. We demonstrate that the conventional PKC (cPKC) isoforms, PKC-alpha, PKC-betaI, and PKC-betaII, as well as RACK1, are expressed in mouse oocytes (germinal vesicle [GV]) and mature eggs (metaphase II [MII]). In GV oocytes, PKC-alpha, PKC-betaII, and RACK1 were uniformly distributed in the cytoplasm, while PKC-betaI was localized in the cytoplasm and in the plasma membrane as well. Treatment of GV oocytes with the biologically active phorbol ester, 12-o-tetradecanoyl phorbol-13-acetate (TPA), resulted in a rapid translocation of the cytosolic PKC-alpha, but not PKC-betaI, PKC-betaII, or RACK1, to the plasma membrane. This was associated with inhibition of GV breakdown. In MII eggs (17 h post-hCG), PKC-alpha was uniformly distributed in the cytoplasm while PKC-betaI and -betaII were distributed in the cytoplasm and in the plasma membrane as well. Treatment with TPA resulted in a rapid translocation of PKC-alpha from the cytoplasm to the plasma membrane and a significant decrease of PKC-betaI throughout the cytoplasm, while it also remained in the cell periphery. No change in the distribution of PKC-betaII or RACK1 was observed. TPA also induced pronucleus formation. Physiological activation of MII eggs by sperm induced cortical granule exocytosis associated with significant translocation of PKC-alpha and -betaI, but not -betaII, to the plasma membrane. Overall, these results suggest a possible involvement of cPKC isoforms in the mechanism of mouse oocyte maturation and egg activation.     

Hormonal regulation of protein kinase C in the mouse mammary gland

The hormonal regulation of protein kinase C (PKC) induction over 3 to 14 days was investigated in the mouse mammary gland in vitro and in vivo. In intact mice, estradiol (1 microgram/mouse injected daily for 2 weeks) stimulated PKC activity 70%, while progesterone (1 mg/mouse injected daily) inhibited it by 30%. Prolactin, whose levels were elevated for 2 weeks by two pituitary isografts, had no effect. When mammary gland explants were cultured in insulin and cortisol, the further addition of estradiol (1 ng/ml), progesterone (1 microgram/ml), or prolactin (1 microgram/ml) did not alter PKC activity after 3 days. These data suggest the following conclusions: although previous studies have implicated prolactin in the transient, calcium-phospholipid activation of PKC, it does not appear to elevate total levels of this kinase over prolonged periods. In contrast, the sex steroids do appear to affect long-term levels of this kinase; furthermore, this latter effect may be indirect.     

Loss of protein kinase C delta alters mammary gland development and apoptosis

As apoptotic pathways are commonly deregulated in breast cancer, exploring how mammary gland cell death is regulated is critical for understanding human disease. We show that primary mammary epithelial cells from protein kinase C delta (PKCδ) −/− mice have a suppressed response to apoptotic agents in vitro. In the mammary gland in vivo, apoptosis is critical for ductal morphogenesis during puberty and involution following lactation. We have explored mammary gland development in the PKCδ −/− mouse during these two critical windows. Branching morphogenesis was altered in 4- to 6-week-old PKCδ −/− mice as indicated by reduced ductal branching; however, apoptosis and proliferation in the terminal end buds was unaltered. Conversely, activation of caspase-3 during involution was delayed in PKCδ −/− mice, but involution proceeded normally. The thymus also undergoes apoptosis in response to physiological signals. A dramatic suppression of caspase-3 activation was observed in the thymus of PKCδ −/− mice treated with irradiation, but not mice treated with dexamethasone, suggesting that there are both target- and tissue-dependent differences in the execution of apoptotic pathways in vivo. These findings highlight a role for PKCδ in both apoptotic and nonapoptotic processes in the mammary gland and underscore the redundancy of apoptotic pathways in vivo.     

Alterations in rat coronary vasoreactivity and vascular protein kinase C isoforms in Type 1 diabetes

Vascular complications associated with diabetes mellitus (DM) have been linked to activation of PKC-dependent signaling pathways in both human and animal models of DM. To determine whether aberrant PKC signaling mechanisms specifically impact the coronary circulation, we assessed isolated coronary artery (CA) responses after the induction of Type 1 DM. Male Sprague-Dawley rats were subjected to partial pancreatectomy (DM; n = 23) and compared with age-matched controls (CTL; n = 19). Vasoreactivity was assessed in single CAs ( approximately 250 microm internal diameter) after abluminal administration of the Gq-dependent vasoconstrictors endothelin (ET)-1 (10(-10)-10(-9) M) and U-44619 (10(-9)-10(-5) M) or the voltage-gated Ca2+ channel agonist BAY K 8644 (10(-9)-10(-5) M) with and without the PKC inhibitor bisindolylmaleimide (Bis; 10(-6) M). Dilator responses to ACh (10(-9)-10(-5) M) were also assessed. ET-1 resulted in significantly greater constriction in the DM versus CTL group (50 +/- 4% vs. 33 +/- 5%, P < 0.0001), whereas responses to U-44619 and BAY K 8644 were similar between groups. Importantly, inhibition of ET-1 and U-44619 constriction by Bis occurred in the DM but not CTL group (P < 0.05). Western blotting on isolated CAs revealed greater levels of PKC-alpha, PKC-beta I, and PKC-beta II by 22%, 15.3%, and 17.6%, respectively, in the DM versus CTL group (P < 0.05), whereas PKC-delta and PKC-epsilon protein levels were unchanged. DM was also associated with attenuated CA dilation after ACh treatment (P < 0.0566) and reductions in endothelial nitric oxide synthase protein levels versus CTL (P < 0.03). These data suggest that Ca2+-dependent PKC signaling pathways, particularly for ET-1, play a greater role in modulating CA vasoconstrictor responses in DM versus CTL. These data further suggest that aberrant CA constrictor and dilator responses are likely to contribute to the coronary vascular pathology associated with DM.     

Novel protein kinase C isoforms and mitogen-activated kinase kinase mediate phorbol ester-induced osteopontin expression

BACKGROUND AND AIMS: The expression of osteopontin (OPN), a protein postulated to play a role in tumorigenesis, is induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) in vivo and in the in vitro initiation-promotion skin carcinogenesis model (JB6 cells). Although TPA-induced OPN expression in JB6 cells has been suggested to involve protein kinase C (PKC), the PKC isoforms and the downstream pathway mediating OPN expression have not been extensively studied. METHODS: Using the JB6 cell model, we determined the involvement of PKC isoforms, mitogen-activated protein kinase kinase (MAPK kinase/MEK) and MAPK in TPA-induced OPN expression using inhibitors specific to PKC isoforms and MEK and performing Northern blot analyses. Western blot analyses of cells treated with specific inhibitors were also performed to determine whether PKC isoforms or MEK were involved in activation of MAPK. KEY RESULTS: TPA increased the steady-state level of OPN mRNA as early as 2-4h and this expression persisted for at least 4 days. TPA induction of OPN expression in JB6 cells is mediated through PKC epsilon and PKC delta, which also mediated the phosphorylation of MAPK. Additionally, inhibition of MEK activity, which activates MAPK, attenuated TPA-induced OPN expression. These findings suggest that activation of MAPK is important in mediating OPN expression. CONCLUSION: TPA-induced steady-state OPN mRNA expression in mouse JB6 cells involves the activation of MAPK mediated through PKC epsilon and/or PKC delta.     

Activation of protein kinase C and cAMP-dependent protein kinase in hormone-dependent mammary gland tumors during stimulation of their growth with progesterone and estrone

GR mouse mammary tumour growth is stimulated by simultaneous administration of progesterone and estrone. These hormones strongly activate cAMP-dependent protein kinases both in the cytosol and in humour cell nuclei by causing the elevation of PK-1 and PK-2 activities. Ovarian hormone action on the proliferation is similar to that of growth factors, i.e., the hormones significantly stimulate the calcium-activated, phospholipid-dependent protein kinase C. Protein kinase C has been discovered is growing tumour cell nuclei. In early periods after ovarian hormone administration protein kinase C is activated in a greater degree as compared to cAMP-dependent protein kinases. A hypothesis on the feasibility of simultaneous activation by steroid hormones of both second messenger systems, namely the cAMP system and the system of production of diacylglycerol from phosphtidylinositol diphosphate is proposed.     

The expression and stage-specific localization of protein kinase C isotypes during mouse preimplantation development

Signaling events mediate many processes that act during embryogenesis to initiate the program of early development. Within the cell many of these changes are mediated through the activation or inactivation of kinases and phosphatases. Protein kinase C (PKC) is one kinase that has been shown to be involved in at least two developmental transitions during early development, fertilization and embryonic compaction. PKC is a family of kinases whose various isotypes have differing requirements for activation of the kinase that include the availability of calcium, diacylglycerol, and negatively charged phospholipids. The presence of more than one isotype in an egg or blastomere of the embryo would provide the possibility that different isotypes mediate distinct signaling pathways in the cells. To address this possibility the different isotypes of PKC were examined at the mRNA and protein levels during preimplantation development in the mouse. Our results demonstrate that seven isotypes of PKC are present during preimplantation development in mouse, some are of maternal origin and others appear after fertilization. Two isotypes have a stage-dependent nuclear localization. In addition, within each blastomere PKC isotypes occupy different subcellular locations in a stage-dependent fashion.     

Alterations in protein kinase C isoenzyme expression and autophosphorylation during the progression of pressure overload-induced left ventricular hypertrophy

Cardiomyocytes express several isoenzymes of protein kinase C (PKC), which as a group have been implicated in the induction of left ventricular hypertrophy (LVH) and its transition to heart failure. Individual PKC isoenzymes also require transphosphorylation and autophosphorylation for enzymatic activity. To determine whether PKC isoenzyme expression and autophosphorylation are altered during LVH progression in vivo, suprarenal abdominal aortic coarctation was performed Sprague-Dawley rats. Quantitative Western blotting was performed on LV tissue 1, 8 and 24 weeks after aortic banding, using antibodies specific for total PKC, PKC and PKC, and their C-terminal autophosphorylation sites. Aortic banding produced sustained hypertension and gradually developing LVH that progressed to diastolic heart failure over time. PKC levels and autophosphorylation were not significantly different from sham-operated controls during any stage of LVH progression. PKC expression levels were also unaffected during the induction of LVH, but increased 3.2 ± 0.8 fold during the transition to heart failure. In addition, there was a high degree of correlation between PKC levels and the degree of LVH in 24 week banded animals. However, autophosphorylated PKC was not increased at any time point. In contrast, PKC autophosphorylation was increased prior to the development of LVH, and also during the transition to heart failure. The increased PKC autophosphorylation in 1 week banded rats was not accompanied by an increase in total PKC, whereas total PKC levels were markedly increased (6.0 ± 1.7 fold) in 24 week banded animals. Furthermore, both phosphorylated and total PKC levels were highly correlated with the degree of LVH in 24 week banded rats. In summary, we provide indirect evidence to indicate that PKC may be involved in the induction of pressure overload LVH, whereas both PKC and PKC may be involved in the transition to heart failure.     

Immunocytochemical expression and localization of protein kinase C in bovine aortic endothelial cells.

Total PKC activity in BAEC incubated for 24 hrs in either 10% serum (FBS) or serum-deprived media (SDM) was similar. However, most of the activity (69%) in the FBS group was detected in the particulate fraction, while it was mainly in the cytosolic fraction (66%) in the SDM group. By confocal microscopy, there was diffuse cytoplasmic localization of the antibodies to the alpha and beta PKC isoforms. gamma PKC was not detected. Treatment of FBS or SDM cells with a phorbol ester resulted in an increase in PKC activity with translocation to the particulate fraction. PKC alpha immunofluorescence redistributed to the perinuclear region whereas PKC beta staining remained mostly cytosolic. Calphostin C, a PKC inhibitor, prevented the phorbol ester-induced increase in PKC activity and translocation.     

Involvement of mu- and m-calpains and protein kinase C isoforms in L8 myoblast differentiation

The objectives were to investigate the roles of different calpains and protein kinase C (PKC) isoforms in muscle differentiation. Concentrations of mu- and m-calpain increased significantly whereas PKCalpha and delta declined significantly during L8 myoblast differentiation. Both mu-calpain and m-calpain antisense oligonucleotides inhibited myotube formation and creatine kinase activity during L8 myoblast differentiation. These results implied that both mu- and m-calpain were involved in L8 myoblast differentiation. To investigate the involvement of calpain in regulation of PKC concentrations, mu-calpain antisense oligonucleotides were added to L8 myoblasts. PKCalpha remained unchanged and PKCdelta declined. By adding m-calpain antisense oligonucleotides instead, PKCalpha level remained unchanged and PKCdelta concentrations increased significantly during differentiation. These results suggest that PKCalpha, but not PKCdelta, is the substrate for mu-calpain and PKCalpha and delta are the substrates for the m-calpain. In addition, more phosphorylated myogenin was found in day 2 antisense oligonucleotides treated L8 cells. It is concluded that the decline of PKCalpha mediated by m- and mu-calpain is essential for L8 myoblast differentiation. The decline of PKC during myoblast differentiation may cause hypo-phosphorylation of myogenin, which in turn activates muscle-specific genes during myogenesis.     

Nuclear protein kinase C isoforms and apoptosis

The process of apoptosis is regulated at multiple levels through phosphorylation by several different protein kinases. The protein kinase C (PKC) family of isozymes have been shown to exert both inhibitory and stimulatory influences on apoptosis. During the apoptotic process phosphorylative events are known to occur also at the nuclear level. Evidence suggests that PKC isoforms play a key role in some steps that lead to nuclear disassembly during the execution phase of apoptosis. This review highlights the recent progress made in determining the roles played by individual PKC nuclear isoforms in the control of apoptosis.     

Alterations in protein kinase C type III-alpha during heat shock of rat embryo fibroblasts.

Heat shock treatment of rat embryo fibroblasts resulted in a 60% increase in cytosolic protein kinase C activity, in contrast to phorbol ester-induced translocation to the membrane. During reversal of the cells back to the normal temperature a decrease in cytosolic PKC activity was observed and paralleled by an increase in protamine kinase activity. Cell lysates prepared from heat shock-treated cells show a marked calcium/phospholipid-dependent phosphorylation of several endogenous PKC substrate proteins, while the 28-kDa stress protein was shown to be a PKC substrate. These cells express the TYPE III-alpha isoform of PKC and, thus, the alterations induced within cells exposed to hyperthermic treatment may reflect a functional significance with regard to the regulation of this specific isoform.     

Comparative expression of endogenous retrotransposons in adult mouse mammary gland during normal development and differentiation

Amplified expression of the endogenous retrotransposons, intracisternal A particles (IAPs) and murine leukemia virus-related elements (MLVEs), along with decreased expression of VL30 elements frequently occurs during mouse mammary tumorigenesis. We have now analyzed the expression of these retroelements during the normal developmental and differentiation cycle of the mammary gland as found in virgin, pregnant, lactating, and post-lactation adult female BALB/c mice. Retrotransposon expression was either unchanged or decreased during the progressive stages of the cycle compared to virgin tissue. Likewise, growth of mammary epithelial cells in primary culture had little or no effect on expression of IAPs, MLVEs and VL30 sequences. Thus, the dramatic changes involving these retrotransposons in many mouse mammary tumors appear unrelated to any normal state.     

Signalling by protein kinase C isoforms in the heart

Understanding transmembrane signalling process is one of the major challenge of the decade. In most tissues, since Fisher and Krebs's discovery in the 1950's, protein phosphorylation has been widely recognized as a key event of this cellular function. Indeed, binding of hormones or neurotransmitters to specific membrane receptors leads to the generation of cytosoluble second messengers which in turn activate a specific protein kinase. Numerous protein kinases have been so far identified and roughly classified into two groups, namely serine/threonine and tyrosine kinases on the basis of the target amino acid although some more recently discovered kinases like MEK (or MAP kinase kinase) phosphorylate both serine and tyrosine residues.Protein kinase C is a serine/threonine kinase that was first described by Takai et al. [1] as a Ca- and phospholipid-dependent protein kinase. Later on, Kuo et al. [2] found that PKC was expressed in most tissues including the heart. The field of investigation became more complicated when it was found that the kinase is not a single molecular entity and that several isoforms exist. At present, 12 PKC isoforms and other PKC-related kinases [3] were identified in mammalian tissues. These are classified into three groups. (1) the Ca-activated -, -,and -PKCs which display a Ca-binding site (C2); (2) the Ca-insensitive -, -, -, -, and -PKCs. The kinases that belong to both of these groups display two cystein-rich domains (C1) which bind phorbol esters (for recent review on PKC structure, see [4]). (3) The third group was named atypical PKCs and include , , and -PKCs that lack both the C2 and one cystein-rich domain. Consequently, these isoforms are Ca-insensitive and cannot be activated by phorbol esters [5]. In the heart. evidence that multiple PKC isoforms exist was first provided by Kosaka et al. [6] who identified by chromatography at least two PKC-related isoenzymes. Numerous studies were thus devoted to the biochemical characterization of these isoenzymes (see [7] for review on cardiac PKCs) as well as to the identification of their substrates.This overview aims at updating the present knowledge on the expression, activation and functions of PKC isoforms in cardiac cells. (Mol Cell Biochem 157: 65–72, 1996)     

Differential expression of protein kinase C isozymes and erythroleukemia cell differentiation

Hexamethylene bisacetamide (HMBA) and other polar/apolar chemical agents are potent inducers of erythroid differentiation in murine erythroleukemia cells (MELC), as well as other transformed cell lines. Although the mechanism of action of HMBA is not yet known, evidence has been obtained that protein kinase C (PKC) plays a role in this process. In this study we provide further evidence that establishes this relationship. MELC contain two principal PKC activities, PKC beta and PKC alpha. MELC variants, selected for resistance to vincristine (VC), which display acceleration of their rates of induced differentiation, are enriched in PKC beta activity. When MELC are exposed to HMBA there is a fall in PKC activity, largely accounted for by a decline in PKC beta. This decline in PKC activity is faster in the VC-resistant, rapidly differentiating MELC. We previously demonstrated that VC-resistant MELC are resistant to the inhibition of differentiation by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). In both VC-sensitive and -resistant MELC, PMA causes rapid membrane translocation and then a decline in PKC activity, accompanied by a generation of a Ca2+- and phospholipid-independent protein kinase activity. In VC/PMA-resistant variants, this Ca2+/phospholipid-independent protein kinase activity persists considerably longer than in the VC-sensitive variants. This correlates with the resistance to PMA and provides additional evidence for a role for the Ca2+/phospholipid-independent protein kinase activity during induced differentiation.     

Analysis of inhibitor of apoptosis protein family expression during mammary gland development

Background  

Inhibitors-of-Apoptosis-Proteins (IAPs) are an evolutionarily conserved family of proteins capable of regulating several facets of apoptosis. IAPs are frequently dysregulated in cancer, but their role in the regulation of apoptosis during developmental processes is not fully understood. Here we examined the expression of IAPs during the post-natal development of the mouse mammary gland, which is a tissue that exhibits a profound induction of apoptosis during involution.     

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.