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During the course of differentiation of preadipocytes into adipocytes, several differentiation‐linked genes are activated synchronously with morphological changes. To follow this process we have used 3T3‐F442A cells, known to undergo adipocyte conversion with high frequency. Accumulation of lipid droplets in the cytoplasm constitutes an easily visualized sign of the terminally differentiated phenotype. In this report we demonstrate that expression of the CCAAT/enhancer binding protein (C/EBP) is an important factor in determining the ability to accumulate lipid droplets in terminally differentiated adipocytes. In one experiment we can suppress C/EBP expression through administration of hydrocortisone to differentiating 3T3‐F442A cells, which is accompanied by an inability of the cells to accumulate lipid. In another experiment a C/EBP antisense expression vector has been stably introduced into 3T3‐F442A cells and as compared with control cells, a 62% decrease of C/EBP mRNA (p less than 0.01) is demonstrated. This decrease of C/EBP mRNA is accompanied by a change in cellular morphology characterized by a reduced ability to form lipid droplets. We can also demonstrate a correlation between the degree of reduction of C/EBP mRNA and the amount of lipid present in the cells. These findings strongly support the view that C/EBP is a necessary component of terminal adipocyte differentiation.

We have isolated and sequenced cDNA clones covering the entire coding sequence of human-milk bile-saltstimulated lipase, as well as 996 nucleotides of the 3' end of the pancreatic enzyme carboxylic ester hydrolase. The deduced amino acid sequence of the lipase starts with a 23-residue leader peptide. The open reading frame continues with 722 amino acid residues. The sequence contains in the C-terminal part a proline-rich repeat, 16 repeats of 11 amino acid residues each. The mRNA was estimated to be approximately 2500 nucleotides from Northern blot and of similar size in mammary and pancreatic tissues. Data obtained indicate that the lipase and the carboxylesterase are identical and coded for by the same gene. The cDNA is 2428 bases long, which indicates that a near full-length copy of the transcript has been isolated. Comparisons with other enzymes show that the lipase is a new member of the supergene family of serine hydrolases. It is not only closely related (and in its Nterminal half virtually identical) to lysophospholipase from rat pancreas and cholesterol esterase from bovine pancreas, but also shows a high degree of similarity to several esterases, e. g. acetylcholine esterase. In contrast, no such similarity could be found to typical lipases.The human lactating mammary gland synthesizes and secretes with the milk a bile-salt-stimulated lipase (BSSL) [l] that, after specific activation by primary bile salts [2], contributes to the breast-fed infant's endogenous capacity for intestinal fat digestion [3-51. This enzyme, which accounts for approximately 1% of total milk protein [6], is a non-specific lipase; in vitro it hydrolyses not only tri-, di-and monoacylglycerols, but also cholesteryl and retinyl esters and lysophosphatidylglycerols unpublished results). Furthermore, its activity is not restricted to emulsified substrates, but micellar and soluble substrates are hydrolyzed at similar rates [I 11.

Progression to metastasis is the proximal cause of most cancer-related mortality. Yet much remains to be understood about what determines the spread of tumor cells. This paper describes a novel pathway in breast cancer that regulates epithelial-to-mesenchymal transition (EMT), motility, and invasiveness. We identify two transcription factors, nuclear factor 1-C2 (NF1-C2) and Forkhead box F1 (FoxF1), downstream of prolactin/ nuclear Janus-activated kinase 2, with opposite effects on these processes. We show that NF1-C2 is lost during mammary tumor progression and is almost invariably absent from lymph node metastases. NF1-C2 levels in primary tumors correlate with better patient survival. Manipulation of NF1-C2 levels by expression of a stabilized version or using small interfering RNA showed that NF1-C2 counteracts EMT, motility, invasiveness, and tumor growth. FoxF1 was found to be a direct repressed target of NF1-C2. We provide the first evidence for a role of FoxF1 in cancer and in the regulation of EMT in cells of epithelial origin. Overexpression of FoxF1 was associated with a mesenchymal phenotype, increased invasiveness in vitro, and enhanced growth of breast carcinoma xenografts in nude mice. The relevance of these findings is strengthened by the correlation between FoxF1 expression and a mesenchymal phenoype in breast cancer cell isolates, consistent with the interpretation that FoxF1 promotes invasion and metastasis. Cancer Res; 70(5); 2020-9. ©2010 AACR.

The classical mechanism by which prolactin transduces its signal in mammary epithelial cells is by activation of cytosolic signal transducer and activator of transcription 5 (Stat5) via a plasma membrane-associated prolactin receptor-Janus kinase 2 (Jak2) complex. Here we describe an alternative pathway through which prolactin via Jak2 localized in the nucleus activates the transcription factor nuclear factor 1-C2 (NF1-C2). Previous reports have demonstrated a nuclear localization of Jak2, but the physiologic importance of nuclear Jak2 has not been clear. We demonstrate that nuclear Jak2 regulates the amount of active NF1-C2 through tyrosine phosphorylation and proteasomal degradation. Our data also demonstrate a link between prolactin and p53 as well as the milk gene carboxyl ester lipase through nuclear Jak2 and NF1-C2. Hence, we describe a novel pathway through which nuclear Jak2 is subject to regulation by prolactin in mammary epithelial cells.The mammary gland is a complex organ that undergoes development and differentiation under the control of a number of hormones and growth factors, their receptors, and transcription factors. The polypeptide hormone prolactin has an essential role both in the differentiation of the gland during pregnancy and in the regulation of milk protein gene expression (13). Prolactin receptor signaling can be mediated through several different signaling pathways (2, 4, 9, 10), the principal of which in mammary epithelial cells is that through the receptor-associated tyrosine kinase Janus kinase 2 (Jak2) (21). Binding of prolactin to its receptor activates Jak2, which phosphorylates the transcription factor signal transducer and activator of transcription 5 (Stat5). Phosphorylated Stat5 dimerizes, translocates to the nucleus, and binds to its response elements (35). Both Jak2 and Stat5 have been demonstrated to be essential for mammary gland development and milk protein gene expression (23,29). Apart from the Jak2-Stat5 pathway, the target genes and factors that mediate the action of prolactin in the mammary gland under normal conditions are poorly understood.One important mechanism regulating signal transduction is that through proteolysis (6, 11). The proteasome pathway in particular plays an important role in the degradation of a number of cellular proteins (26,31). Earlier studies have shown that proteasome inhibitors prolong the activity of the Jak-Stat signaling pathway by protecting tyrosine-phosphorylated Jak2 proteins from degradation (33, 38).We have previously identified the transcription factor nuclear factor 1-C2 (NF1-C2) as an important activator of milk genes as well as the p53 tumor suppressor gene in the mouse mammary gland during pregnancy (14,16). This activity indicates that NF1-C2 might participate both in the establishment of a functional gland and in the protection of the gland against tumorigenesis during proliferation. Further, using studies with mouse mammary epithelial NMuMG cells and mammary tissue from heterozygous prolactin receptor knockout mice as a b...

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Tumour necrosis factor (TNF) was previously shown to suppress lipoprotein lipase (LPL) synthesis and activity in 3T3-L1 adipocytes. The present study examined the effect of TNF on amounts of mRNA for LPL in 3T3-L1 cells. Northern-blot analysis of polyadenylated RNA using a cDNA probe to guinea-pig LPL identified two predominant species of LPL message, 3.7 and 3.9 kilobases in size. The steady-state amounts of these mRNAs increased 10-fold upon expression of the adipocyte phenotype. A single dose of 1.5 nM-TNF decreased LPL mRNA by approx. 60% in 17 h with a corresponding decrease in LPL activity, an effect that was reversed 48 h after exposure to TNF. The results demonstrate that TNF reversibly down-regulates LPL mRNA in fully differentiated 3T3-L1 adipocytes. Cells induced to differentiate in the presence of 1.5 nM-TNF exhibited a delayed time course for development of the adipocyte phenotype, as judged by attenuation of the normal increase in LPL mRNA that occurs with differentiation.