Itamar Barash

The Biological Functions of the Versatile Transcription Factors STAT3 and STAT5 and New Strategies for their Targeted Inhibition

Signal transducers and activators of transcription (STATs) comprise a unique family of transcription factors, which transmit the interactions of cytokines, hormones and growth factors with their cell surface receptors into transcriptional programs. The mechanism of STAT activation has been well-established and comprises tyrosine phosphorylation, dimerization, nuclear translocation, binding to specific DNA response elements, recruitment of co-activators or co-repressors and transcriptional induction or repression of target genes. Gene deletion, microarrays, proteomics and chromatin immunoprecipitation experiments have revealed target genes with a broad range of functions regulated by STAT3 and STAT5. In the mammary gland, STAT5-induced genes contribute mainly to the prolactin dependent lobulo-alveolar development, whereas STAT3 induced genes control apoptosis during involution. Crucial effects have also been observed in other tissues. The germ line deletion of STAT3 or STAT5 causes early embryonal or perinatal lethality in mice. STAT5 is also required for proliferation of T- and B-cells and hematopoietic stem cell self-renewal. Deregulated STAT activity is often found associated with tumorigenesis and activated STATs seem to be limiting components in tumor cells. This review summarizes the functions of STAT3 and STAT5 in different cell types and the strategies that are used to counteract their action in tumor cells.

Expression of human serum albumin in the milk of transgenic mice

We have tested the feasibility of producing large quantities of human serum albumin (HSA) in the milk of transgenic livestock by generating transgenic mice as a model system. The sheep β-lactoglobulin (BLG) 5′-regulatory promoter sequences were used to support expression of BLG or HSA in transgenic mice. Transgenic animals generated from the entire BLG gene including 3, 5.5 or 10.8 kb of 5′-sequences demonstrated that 3 kb of 5′-sequences were sufficient to support high levels of expression of BLG, and that the longer 5′-sequences did not improve upon the levels of expression. As such, the 3 kb 5′-sequences were used to drive expression of HSA in BLG-HSA constructs. HSA was not detectably expressed in eight transgenic lines generated from a BLG-HSA construct containing the HSA cDNA. Two transgenic lines of 26 generated, using five different constructs, with an HSA minigene possessing the first intron expressed HSA in their milk. One of these expressed HSA at high levels (2.5 mg ml−1) and has stably transmitted this ability to its progeny. A high percentage of transgenic mouse lines (four of six) generated from a vector containing an HSA minigene possessing introns 1 and 2 expressed HSA in their milk at levels which ranged from 1 to 35 μg ml−1. In a similar trend, levels of expression of HSA by transfected tissue culture cells from BLG-HSA vectors containing an introduced SV40 enhancer were low with the HSA cDNA, increased with the HSA minigene with intron 1 and increased further with the minigene containing introns 1 and 2. This study demonstrates that high levels of HSA can be expressed in the milk of transgenic animals, that introns of the HSA gene play a role in its expression and that transfected cell lines may be used to quickly evaluate the relative expression efficiencies of various vector constructs intended for future transgenic evaluation.

Deregulation of Stat5 expression and activation causes mammary tumors in transgenic mice

Members of the signal transducers and activators of transcription (Stat) family regulate essential cellular growth and survival functions in normal cells and have also been implicated in tumorigenesis. We have studied the potential role of Stat5 in mammary tumorigenesis by targeting Stat5 variants to the mammary gland of transgenic mice using regulatory sequences of the β‐lactoglobulin gene. Mammary‐directed expression of the wild‐type Stat5, constitutively activated Stat5 and carboxyl‐terminally truncated dominant negative Stat5 forms resulted in mammary tumors with incidence rates of up to 22% and latency periods of 8–12 months. Undifferentiated carcinomas most frequently occurred in mice expressing the carboxyl‐terminally truncated Stat5. The more differentiated papillary and micropapillary adenocarcinomas were primarily found in mice overexpressing the native and constitutively active transgenes. Higher levels of translation initiation factor 4E (eIF4E) and cyclin D1 expression but lower levels of activated Stat3 were found in tumors of mice expressing the constitutively active Stat5 when compared to mice expressing the wild‐type or truncated forms. A higher expression of the estrogen receptor (ERα) was observed in carcinomas compared to other phenotypes. The ability of both forms of Stat5, the transactivating form and the dominant negative form, to participate in oncogenesis indicates that there is more than one mechanism by which Stat5 contributes to this process. The transactivation function of Stat5 is involved in the determination of tumors with a more differentiated phenotype.

Role of amino acids in translational mechanisms governing milk protein synthesis in murine and ruminant mammary epithelial cells

The role of amino acids (AA) on translational regulation in mammary epithelial cells cultured under lactogenic conditions was studied. The rates of total protein synthesis and β‐lactoglobulin (BLG) synthesis in mouse CID‐9 cells were 2.1‐ or 3.1‐fold higher, respectively, than in their bovine L‐1 counterparts. Total AA deprivation or selective deprivation of Leu had a negative protein‐specific effect on BLG synthesis that was more pronounced in bovine cells than in murine cells. Dephosphorylation of eukaryotic initiation factor 4E‐binding protein 1 (4E‐BP1) and S6 kinase (S6K1) on Thr389 but not on Ser411 was also more prominent in bovine cells. Noteably, deprivation of Leu had a less marked effect on BLG synthesis and 4E‐BP1 or S6K1 phosphorylation than deprivation of all AA. In AA‐deprived CID‐9 cells, Leu specifically restored BLG synthesis from pre‐existing mRNA whereas AA also restored total protein synthesis. This restoration was associated with a more pronounced effect on 4E‐BP1 and S6K1 phosphorylation in bovine versus murine cells. Rapamycin specifically reduced Leu‐ and AA‐stimulated BLG translation initiation in a dose‐dependent manner. A further reduction was observed for Leu‐treated cells in the presence of LY294002, a PI3K (phosphatidylinositol 3‐kinase) inhibitor, which also reduced total protein synthesis. These findings suggest that direct signaling from AA to the translational machinery is involved in determining the rates of milk protein synthesis in mammary epithelial cells. J. Cell. Biochem. 98: 685–700, 2006.

Characterization of an epithelial cell line from bovine mammary gland.

SummaryElucidation of the bovine mammary glands unique characteristics depends on obtaining an authentic cell line that will reproduce its function in vitro. Representative clones from bovine mammary cell populations, differing in their attachment capabilities, were cultured. L-1 cells showed strong attachment to the plate, whereas H-7 cells detached easily. Cultures established from these clones were nontumorigenic upon transplantation to an immunodeficient host; they exhibited the epithelial cell characteristics of positive cytokeratin but not smooth muscle actin staining. Both cell lines depended on fetal calf serum for proliferation. They exhibited distinct levels of differentiation on Matrigel in serum-free, insulin-supplemented medium on the basis of their organization and β-lactoglobulin (BLG) secretion. H-7 cells organized into mammospheres, whereas L-1 cells arrested in a duct-like morphology. In both cell lines, prolactin activated phosphorylation of the signal transducer and activator of transcription, Stat5—a regulator of milk protein gene transcription, and of PHAS-I—an inhibitor of translation initiation in its nonphosphorylated form. De novo synthesis and secretion of BLG were detected in differentiated cultures: in L-1 cells, BLG was dependent on lactogenic hormones for maximal induction but was less stringently controlled than was β-casein in the mouse CID-9 cell line. L-1 cells also encompassed a near-diploid chromosomal karyotype and may serve as a tool for studying functional characteristics of the bovine mammary gland.

Ectopic expression of β-lactoglobulin/human serum albumin fusion genes in transgenic mice: hormonal regulation andin situ localization

We produced transgenic mice carrying the native sheep β-lactoglobulin (BLG) or fusion genes composed of the BLG promoter and human serum albumin (HSA) minigenes. BLG was expressed exclusively in the mammary glands of the virgin and lactating transgenic mice evaluated. In contrast, transgenic females carrying the BLG/HSA fusion constructs also expressed the HSA RNA ectopically in skeletal muscle, kidney, brain, spleen, salivary gland and skin. Ectopic expression of HSA RNA was detected only in strains that express the transgene in the mammary gland. There was no obvious correlation between the level of the HSA RNA expressed in the mammary gland and that found ectopically. In three transgenic strains analysed, the expression of HSA RNA in kidney and skeletal muscle increased during pregnancy and lactation, whereas in the brain HSA expression decreased during lactation in one of the strains. HSA protein was synthesized in skeletal muscle and skin of strain #23 and its level was higher in lactating mice compared with virgin mice. Expression of HSA was also analysed in males and was found to be more stringently controlled than in females of the same strains.In situ hybridization analyses localized the expressed transgene in the skin, kidney, brain and salivary glands of various transgenic strains. Distinct strain-specific and cell-type specific HSA expression patterns were observed in the skin. This is in contrast to the exclusive expression of the HSA transgene in epithelial cells surrounding the alveoli of the mammary gland. Taken together, these results suggest that the absence of sufficient mammary-specific regulatory elements in the BLG promoter sequences and/or the juxtaposition of the BLG promoter with the HSA coding sequences leads to novel tissue- and cell-specific expression in ectopic tissues of transgenic mice.

Cell Hierarchy and Lineage Commitment in the Bovine Mammary Gland

The bovine mammary gland is a favorable organ for studying mammary cell hierarchy due to its robust milk-production capabilities that reflect the adaptation of its cell populations to extensive expansion and differentiation. It also shares basic characteristics with the human breast, and identification of its cell composition may broaden our understanding of the diversity in cell hierarchy among mammals. Here, Lin− epithelial cells were sorted according to expression of CD24 and CD49f into four populations: CD24medCD49fpos (putative stem cells, puStm), CD24negCD49fpos (Basal), CD24highCD49fneg (putative progenitors, puPgt) and CD24medCD49fneg (luminal, Lum). These populations maintained differential gene expression of lineage markers and markers of stem cells and luminal progenitors. Of note was the high expression of Stat5a in the puPgt cells, and of Notch1, Delta1, Jagged1 and Hey1 in the puStm and Basal populations. Cultured puStm and Basal cells formed lineage-restricted basal or luminal clones and after re-sorting, colonies that preserved a duct-like alignment of epithelial layers. In contrast, puPgt and Lum cells generated only luminal clones and unorganized colonies. Under non-adherent culture conditions, the puPgt and puStm populations generated significantly more floating colonies. The increase in cell number during culture provides a measure of propagation potential, which was highest for the puStm cells. Taken together, these analyses position puStm cells at the top of the cell hierarchy and denote the presence of both bi-potent and luminally restricted progenitors. In addition, a population of differentiated luminal cells was marked. Finally, combining ALDH activity with cell-surface marker analyses defined a small subpopulation that is potentially stem cell- enriched.

Specific combinations of human serum albumin introns direct high level expression of albumin in transfected COS cells and in the milk of transgenic mice

A new series of expression vectors, each comprised of the β-lactoglobulin (BLG) promoter driving one of a variety of human serum albumin (HSA) minigenes or the entire gene, were evaluated for their ability to direct expression of HSAin vitro in COS tissue culture cells and into the milk of transgenic mice. Vectors directed a hierarchy of expression levelsin vitro, dependent upon the specific complement of HSA introns included. HSA introns acted in a synergistic manner. In addition, minigenes comprised of specific subsets of introns were more efficacious than the entire HSA gene with all of its introns. Transgenic mice expressed as much as 10 mg ml−1 of HSA in their milk. Vectors comprised of specific intron subsets directed levels at 1 mg ml−1 or greater in the milk of 20% of generated transgenics. A statistical correlation between the expression level trendin vitro with the trend of expressionin vivo (% which express) at detectable levels (p=0.0015) and at the level of greater than 0.1 mg ml−1 (p=0.0156) was demonstrated. A weak correlation existed (p=0.0526) atin vivo levels of 1 mg ml−1 or greater. These new vectors are expected to direct the production of high levels of HSA in the milk of a large percentage of generated transgenic dairy animals.

Negative effects of the amino acids Lys, His, and Thr on S6K1 phosphorylation in mammary epithelial cells

The role of essential amino acids (AA) on protein synthesis via the mTOR pathway was studied in murine mammary epithelial cells cultured under lactogenic conditions. Leu, Ile, and Val increased S6K1 phosphorylation compared to that measured in AA‐deprived cells. Trp, Phe, and Met had no effect. Surprisingly, Lys, His, and Thr inhibited S6K1 phosphorylation in both murine and bovine mammary cells. Thr exhibited the most potent inhibition, being the only amino acid that competed with Leus positive role. In non‐deprived cells, there was no observable effect of Lys, His, or Thr on S6K1 phosphorylation at concentrations up to five times those in the medium. However, their addition as a mix revealed a synergistic negative effect. Supplementation of Lys, His, and Thr abrogated mTOR Ser 2448 phosphorylation, with no effect on Akt Ser 473—an mTORC2 target. This confirms specific mTORC1 regulation of S6K1 phosphorylation. The individual supplementation of Lys, His, and Thr maintained a low level of IRS‐1 phosphorylation, which was dose‐dependently increased by their combined addition. Thus, in parallel to inhibiting S6K1 activity, these AA may act synergistically to activate an additional kinase, phosphorylating IRS‐1 via an S6K1‐independent pathway. In cultures supplemented by Lys, His, and Thr, cellular protein synthesis decreased by up to 65%. A more pronounced effect was observed on β‐casein synthesis. These findings indicate that positive and negative signaling from AA to the mTOR pathway, combined with modulation of insulin sensitization, mediate the synthesis rates of total and specific milk proteins in mammary epithelial cells. J. Cell. Biochem. 105: 1038–1047, 2008.

Synthesis and secretion of human serum albumin by mammary gland explants of virgin and lactating transgenic mice

Transgenic mice were produced, carrying hybrid genes comprised of the ovine β-lactoglobulin (BLG) milk protein gene promoter and human serum albumin (HSA) coding sequences.In situ hybridization revealed high levels of BLG/HSA hybrid mRNA, confined to the epithelial cells of the lactating mammary gland with a several hundred fold lower concentration in virgin mammary glands. During the first 24 h in culture, exceptionally high levels of HSA were secreted from explants of virgin mice, independent of hormonal control. HSA secretion was reduced considerably during subsequent days in culture and became dependent on the presence of insulin, hydrocortisone and prolactin. This temporal and hormonal pattern of regulation of HSA was different than that found for the secretion of caseins. In contrast to the vast difference in the mRNA content, the amount of HSA secreted from explants derived from lactating mice during the first 24 h in culture was only 2-to 5-fold higher than that found with explants from virgin transgenic mice, suggesting post-transcriptional control of HSA synthesis. The high-level synthesis and secretion of HSA in mammary explants of lactating mice was also dependent on the presence of insulin, hydrocortisone and prolactin. This study confirms previous suggestion that mammary explants from virgin transgenics may serve as a powerful tool for screening the potential of transgenic animals to secrete foreign proteins in their milk.