The role of Stat3 in apoptosis and mammary gland involution. Conditional deletion of Stat3.

STATs (signal transducer and activator of transcription) are a family of latent transcription factors which are activated in response to a variety of cytokines and growth factors. This family of signalling molecules have been implicated in growth, differentiation, survival and apoptosis. In this article, we will review work which highlights the role of individual STAT factors in mammary gland and demonstrate the value of genetically modified mice in defining the function of STAT3. Involution of the mouse mammary gland is characterised by extensive apoptosis of the epithelial cells and the activation of STAT3. STATs 3 and 5 have reciprocal patterns of activation throughout a mammary developmental cycle suggesting that STAT5 may be a survival factor and STAT3 a death factor for differentiated mammary epithelium. To clarify the role of STAT3 in mammary epithelial apoptosis, we have generated a conditional knockout using the lox/Cre recombination system. Mammary glands from crosses of transgenic mice expressing Cre recombinase under the control of the beta-lactoglobulin milk protein gene promoter with mice harbouring one floxed STAT3 allele and one null STAT3 allele, showed a decrease in epithelial apoptosis and a dramatic delay of the involution process upon forced weaning. This was accompanied by precocious activation of STAT1 and increases in p53 and p21 levels--these may act as a compensatory mechanism for initiating the eventual involution which occurs in STAT3 null mammary glands. This demonstrates for the first time the importance of STAT factors in signalling the initiation of physiological apoptosis in vivo and highlights the utility of the lox/Cre system for addressing the function of genes, which have an embryonic lethal phenotype, specifically in mammary gland.

Selection of ventricular-like cardiomyocytes from ES cells in vitro.

Ischemic disorders of the heart can cause an irreversible loss of cardiomyocytes resulting in a substantial decrease of cardiac output. The therapy of choice is heart transplantation, a technique that is hampered by the low number of donor organs. In the present study, we describe the specific labeling, rapid but gentle purification and characterization of cardiomyocytes derived from mouse pluripotent embryonic stem (ES) cells. To isolate the subpopulation of ventricular-like cardiomyocytes, ES cells were stable transfected with the enhanced green fluorescent protein (EGFP) under transcriptional control of the ventricular-specific 2.1 kb myosin light chain-2v (MLC-2v) promoter and the 0.5 kb enhancer element of the cytomegalovirus (CMV(enh).). First fluorescent cells were detected at day 6 + 8 of differentiation within EBs. Four weeks after initiation of differentiation 25% of the cardiomyocyte population displayed fluorescence. Immunohistochemistry revealed the exclusive cardiomyogenic nature of EGFP-positive cells. This was further corroborated by electrophysiological studies where preferentially ventricular phenotypes, but no pacemaker-like cardiomyocytes, were detected among the EGFP-positive population. The enzymatic digestion of EBs, followed by Percoll gradient centrifugation and fluorescence-activated cell sorting, resulted in a 97% pure population of cardiomyocytes. Based on this study, ventricular-like cardiomyocytes can be generated in vitro from EBs and labeled using CMV(enh)./MLC-2v-driven marker genes facilitating an efficient purification. This method may become an important tool for future cell replacement therapy of ischemic cardiomyopathy especially after the proof of somatic differentiation of human ES cells in vitro.

Suppression of epithelial apoptosis and delayed mammary gland involution in mice with a conditional knockout of Stat3.

Mammary gland involution is characterized by extensive apoptosis of the epithelial cells. At the onset of involution, Stat3 is specifically activated. To address the function of this signaling molecule in mammary epithelial apoptosis, we have generated a conditional knockout of Stat3 using the Cre-lox recombination system. Following weaning, a decrease in apoptosis and a dramatic delay of involution occurred in Stat3 null mammary tissue. Involution is normally associated with a significant increase in IGFBP-5 levels. This was observed in control glands, but not in the absence of Stat3. IGFBP-5 has been suggested to induce apoptosis by sequestering IGF-1 to casein micelles, thereby inhibiting its survival function. Our findings suggest that IGFBP-5 is a direct or indirect target for Stat3 and its upregulation is essential to normal involution. No marked differences were seen in the regulation of Stat5, Bcl-x(L), or Bax in the absence of Stat3. Precocious activation of Stat1 and increases in levels of p53 and p21 occurred and may act as compensatory mechanisms for the eventual initiation of involution observed in Stat3 null mammary glands. This is the first demonstration of the importance of a Stat factor in signaling the initiation of physiological apoptosis in vivo.

Efficient BLG-Cre mediated gene deletion in the mammary gland.

Using the phage P1-derived Cre/loxP recombination system, we have developed a strategy for efficient mammary tissue specific inactivation of floxed genes. Transgenic mice were generated which express Cre DNA-recombinase under the control of the mammary gland specific promoter of the ovine beta-lactoglobulin (BLG) gene. To test the specificity of Cre mediated recombination, we crossed these mice to animals harbouring a floxed DNA ligase I allele. We show that the BLG-Cre construct specifies mammary specific gene deletion, and furthermore that it is temporally regulated, predominantly occurring during lactation. We fully characterised the extent of gene deletion in one line (line 74). In this strain the virgin gland is characterised by low levels (7%) of Cre mediated deletion, whereas 70-80% of cells within the lactating mammary gland have undergone recombination. Immunohistochemistry and indirect in situ PCR were used respectively to demonstrate that both Cre protein and Cre activity were evenly distributed throughout the population of secretory epithelial cells. The level of background recombination in non-mammary tissues was found to be < or = 1.1%, irrespective of mammary gland developmental status. Crossing the transgenic BLG-Cre strain described here to mice harbouring other floxed alleles will facilitate the functional analysis of those genes during differentiation and development of the mammary gland.

Annexin VII relocalization as a result of dystrophin deficiency.

Annexin VII (synexin) is a member of the annexin family of proteins, which are characterized by Ca(2+)-dependent binding to phospholipids. In normal skeletal muscle annexin VII is located preferentially at the plasma membrane and the t-tubule system [Selbert et al. (1995) J. Cell. Sci. 108, 85-95]. Here we have analyzed the distribution of annexin VII in muscle disorders in which the Ca2+ regulation is affected. A remarkable difference was observed in muscle specimens from patients suffering from Duchenne muscular dystrophy and also in muscle from the MDX mouse where annexin VII was gradually released from the sarcolemmal membrane into the cytosol and into the extracellular space during progression of the disease. Hypercontracted muscle fibers positive in Ca2+ staining were devoid of cytosolic annexin VII. Annexins IV and VI were similarly released into the extracellular space. Whereas normal skeletal muscle showed specifically the 51-kDa annexin VII isoform, in dystrophic muscle different ratios of the 51-kDa and the muscle-atypic 47-kDa isoforms were observed. The potential of annexin VII to serve as a tool with which cellular Ca2+ levels can be studied and different muscular disorders classified is discussed.

Expression and localization of annexin VII (synexin) in muscle cells.

Annexin VII (synexin) is a member of the annexin family of proteins, which are characterized by Ca(2+)-dependent binding to phospholipids. We used PCR to isolate from a lambda gt11-mouse fibroblast library annexin VII cDNA fragments corresponding to the two isoforms found in both humans and Dictyostelium discoideum. The two isoforms of 47 kDa and 51 kDa differed by 22 amino acids inserted into the proximal third of the hydrophobic N terminus. Annexin VII-specific polypeptides expressed in Escherichia coli were used to generate isoform-specific monoclonal antibodies. Expression of the two isoforms during myogenesis was followed in the myogenic cell lines BC3H1 and L6. Only the 47 kDa isoform was present in undifferentiated L6 or BC3H1 myoblasts. The 51 kDa isoform appeared after myogenesis had been induced and in striated muscle only the 51 kDa isoform was observed. Immunofluorescence showed that annexin VII was located in the cytosol of mononucleated and fused polynucleated cultured cells, whereas in striated muscle, annexin VII was located preferentially at the plasma membrane and the transverse tubules. However, there was also some residual cytosolic staining, which was more abundant in type II (fast twitch) than in type I (slow twitch) fibers. Permeabilization of L6 cells with digitonin in the presence of 5 mM EGTA led to a release of annexin VII from the cells, which paralleled the loss of cytosolic lactate dehydrogenase (LDH) at low detergent concentrations (50 microM). In the presence of 100 microM extracellular Ca2+, annexin VII remained bound to the plasma membrane even in the presence of high digitonin concentrations. Incubation with the Ca(2+)-specific ionophore A23187 and 100 microM extracellular Ca2+ led to a redistribution of annexin VII from the cytosol to the plasma membrane after 30 minutes of incubation. The results obtained indicate a developmentally and Ca(2+)-regulated localization and expression of annexin VII and raise the possibility that annexin VII may play a role in excitation-contraction coupling in skeletal muscle.