The molecular components which mediate cytokine signaling from the cell membrane to the nucleus were studied. Upon the interaction of cytokines with their receptors, members of the janus kinase (Jak) family of cytoplasmic protein tyrosine kinases and of the signal transducers and activators of transcription (Stat) family of transcription factors are activated through tyrosine phosphorylation. It has been suggested that the Stat proteins are substrates of the Jak protein tyrosine kinases. MGF‐Stat5 is a member of the Stat family which has been found to confer the prolactin response. MGF‐Stat5 can be phosphorylated and activated in its DNA binding activity by Jak2. The activation of MGF‐Stat5 is not restricted to prolactin. Erythropoietin (EPO) and growth hormone (GH) stimulate the DNA binding activity of MGF‐Stat5 in COS cells transfected with vectors encoding EPO receptor and MGF‐Stat5 or vectors encoding GH receptor and MGF‐Stat5. The activation of DNA binding by prolactin, EPO and GH requires the phosphorylation of tyrosine residue 694 of MGF‐Stat5. The transcriptional induction of a beta‐casein promoter luciferase construct in transiently transfected COS cells is specific for the prolactin activation of MGF‐Stat5; it is not observed in EPO‐ and GH‐treated cells. In the UT7 human hematopoietic cell line, EPO and granulocyte‐macrophage colony stimulating factor activate the DNA binding activity of a factor closely related to MGF‐Stat5 with respect to its immunological reactivity, DNA binding specificity and molecular weight. These results suggest that MGF‐Stat5 regulates physiological processes in mammary epithelial cells, as well as in hematopoietic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro, mesenchymal stem cells differentiate to osteoblasts when exposed to bone-inducing medium. However, adipocytes are also formed. We showed that activation of the nuclear protein deacetylase Sirt1 reduces adipocyte formation and promotes osteoblast differentiation.Introduction: Mesenchymal stem cells (MSCs) can differentiate into osteoblasts, adipocytes, chondrocytes, and myoblasts. It has been suggested that a reciprocal relationship exists between the differentiation of MSCs into osteoblasts and adipocytes. Peroxisome proliferator-activated receptor ␥2 (PPAR␥2) is a key element for the differentiation into adipocytes. Activation of Sirt1 has recently been shown to decrease adipocyte development from preadipocytes through inhibition of PPAR␥2. Materials and Methods:We used the mouse mesenchymal cell line C3H10T1/2 and primary rat bone marrow cells cultured in osteoblast differentiation medium with or without reagents affecting Sirt1 activity. Adipocyte levels were analyzed by light microscopy and flow cytometry (FACS) after staining with Oil red O and Nile red, respectively. Osteoblast and adipocyte markers were studied with quantitative real-time PCR. Mineralization in cultures of primary rat bone marrow stromal cells was studied by von Kossa and alizarin red staining. Results: We found that Sirt1 is expressed in the mesenchymal cell line C3H10T1/2. Treatment with the plant polyphenol resveratrol as well as isonicotinamide, both of which activate Sirt1, blocked adipocyte development and increased the expression of osteoblast markers. Nicotinamide, which inhibits Sirt1, increased adipocyte number and increased expression of adipocyte markers. Furthermore, activation of Sirt1 prevented the increase in adipocytes caused by the PPAR␥-agonist troglitazone. Finally, activation of Sirt1 in rat primary bone marrow stromal cells increased expression of osteoblast markers and also mineralization. Conclusions: In this study, we targeted Sirt1 to control adipocyte development during differentiation of MSCs into osteoblasts. The finding that resveratrol and isonicotinamide markedly inhibited adipocyte and promoted osteoblast differentiation may be relevant in the search for new treatment regimens of osteoporosis but also important for the evolving field of cell-based tissue engineering.

Estrogen is essential for growth and development of the mammary glands and has been associated with the promotion and growth of breast cancer and in line with this, most human breast cancers are initially estrogen-dependent and undergo regression when deprived of their supporting hormone. Estrogen exerts many of its effects via two nuclear estrogen receptors (ERs), ERα and ERβ. The discovery of a second ER, ERβ, demanded a full re-evaluation of estrogen action in all target tissues and different estrogen associated diseases, including human breast cancer. However, despite over 15 years of research, the exact role, if any, of ERβ in human breast cancer remains elusive. The main challenges now are to develop highly selective anti-ERβ antibodies that are applied to large well characterized human breast cancer samples to validate their diagnostic potential and to explore ERβ-selective agonists in animal models of breast cancer to validate their therapeutic potential.

Mesenchymal stem cells (MSC) can differentiate into osteoblasts, adipocytes, chondrocytes and myoblasts. It has been suggested that a reciprocal relationship exists between the differentiation of MSC into osteoblasts and adipocytes. Peroxisome proliferator-activated receptor γ2 (PPARγ2) is a key element for the differentiation into adipocytes. Activation of the nuclear protein deacetylase Sirt1 has recently been shown to decrease adipocyte development from preadipocytes via inhibition of PPARγ2. In vitro, MSC differentiate to osteoblasts when exposed to bone-inducing medium. However, adipocytes are also developed. In the present study we have targeted Sirt1 to control adipocyte development during differentiation of MSC into osteoblasts. The finding that resveratrol and isonicotinamide markedly inhibited adipocyte and promoted osteoblast differentiation demonstrates an interesting alternative to PPARγ antagonists. These results are important for the evolving field of cell-based tissue engineering, but may also be relevant in the search for new treatments of osteoporosis.

Previous observations have shown that binding of growth hormone to its receptor leads to activation of transcription factors via a mechanism involving phosphorylation on tyrosine residues. In order to establish whether the prolactin-activated transcription factor Stat 5 (mammary gland factor) is also activated by growth hormone, nuclear extracts were prepared from COS-7 cells transiently expressing transfected Stat 5 and growth hormone receptor cDNA. Gel electrophoresis mobility shift analyses revealed the growth hormone-dependent presence of specific DNA-binding proteins in these extracts. The complexes formed could be supershifted by polyclonal anti-Stat 5 antiserum. In other experiments nuclear extracts from growth hormone-treated Chinese hamster ovary cells stably expressing transfected growth hormone receptor cDNA and liver from growth hormone-treated hypophysectomized rats were used for gel electrophoresis mobility shift analyses. These also revealed the presence of specific DNA-binding proteins sharing antigenic determinants with Stat 5. Stat 5 cDNA was shown to be capable of complementing the growth hormone-dependent activation of transcription of a reporter gene in the otherwise unresponsive COS-7 cell line. This complementation was dependent on the presence of Stat 5 tyrosine 694, suggesting a role for phosphorylation of this residue in growth hormone-dependent activation of DNA-binding and transcription.