Advertisement

Endocrine

, Volume 46, Issue 3, pp 568–576 | Cite as

Knockdown of prolactin receptors in a pancreatic beta cell line: effects on DNA synthesis, apoptosis, and gene expression

  • Ramamani ArumugamEmail author
  • Don Fleenor
  • Michael FreemarkEmail author
Original Article

Abstract

Prolactin (PRL) and placental lactogen stimulate beta cell replication and insulin production in vitro and in vivo. The molecular mechanisms by which lactogens promote beta cell expansion are unclear. We treated rat insulinoma cells with a PRL receptor (PRLR) siRNA to determine if PRLR signaling is required for beta cell DNA synthesis and cell survival and to identify beta cell cycle genes whose expression depends upon lactogen action. Effects of PRLR knockdown were compared with those of PRL treatment. PRLR knockdown (−80 %) reduced DNA synthesis, increased apoptosis, and inhibited expression of cyclins D2 and B2, IRS-2, Tph1, and the anti-apoptotic protein PTTG1; p21 and BCL6 mRNAs increased. Conversely, PRL treatment increased DNA synthesis, reduced apoptosis, and enhanced expression of A, B and D2 cyclins, CDK1, IRS-2, FoxM1, BCLxL, and PTTG1; BCL6 declined. PRLR signaling is required for DNA synthesis and survival of rat insulinoma cells. The effects of lactogens are mediated by down-regulation of cell cycle inhibitors (BCL6, p21) and induction of A, B, and D2 cyclins, IRS-2, Tph1, FoxM1, and the anti-apoptotic proteins BCLxL and PTTG1.

Keywords

Lactogen Cyclin BCL6 PTTG1 IRS-2 FoxM1 

Notes

Acknowledgments

The authors thank the National Hormone & Peptide program and Dr. A.F. Parlow, Scientific Director for providing rat PRL. These studies were supported by grants from the NICHD (HD024192) and the American Diabetes Association (7-08-RA-46) (to MF), the Duke Children’s Miracle Network (to RA), and Duke’s Neonatal Perinatal Research Institute.

References

  1. 1.
    P.C. Butler, J.J. Meier, A.E. Butler, A. Bhushan, The replication of beta cells in normal physiology, in disease and for therapy. Nat. Clin. Pract. Endocrinol. Metab. 3, 758–768 (2007)PubMedCrossRefGoogle Scholar
  2. 2.
    J.J. Meier, A.E. Butler, Y. Saisho et al., Beta-cell replication is the primary mechanism subserving the postnatal expansion of beta-cell mass in humans. Diabetes 57, 1584–1594 (2008)PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    F.A. Van Assche, Quantitative morphologic and histoenzymatic study of the endocrine pancreas in nonpregnant and pregnant rats. Am. J. Obstet. Gynecol. 118, 39–41 (1974)PubMedGoogle Scholar
  4. 4.
    A.E. Butler, L. Cao-Minh, R. Galasso et al., Adaptive changes in pancreatic beta cell fractional area and beta cell turnover in human pregnancy. Diabetologia 53, 2167–2176 (2010)PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    P.H. Whincup, S.J. Kaye, C.G. Owen et al., Birth weight and risk of type 2 diabetes: a systematic review. JAMA 300, 2886–2897 (2008)PubMedCrossRefGoogle Scholar
  6. 6.
    M. Freemark, Placental hormones and the control of fetal growth. J. Clin. Endocrinol. Metab. 95, 2054–2057 (2010)PubMedCrossRefGoogle Scholar
  7. 7.
    D. Newbern, M. Freemark, Placental hormones and the control of maternal metabolism and fetal growth. Curr. Opin. Endocrinol. Diabetes Obes. 18, 409–416 (2011)PubMedCrossRefGoogle Scholar
  8. 8.
    M. Freemark, Regulation of maternal metabolism by pituitary and placental hormones: roles in fetal development and metabolic programming. Horm. Res. 65(Suppl 3), 41–49 (2006)PubMedCrossRefGoogle Scholar
  9. 9.
    M. Freemark, K. Kirk, C. Pihoker, M.C. Robertson, R.P. Shiu, P. Driscoll, Pregnancy lactogens in the rat conceptus and fetus: circulating levels, distribution of binding, and expression of receptor messenger ribonucleic acid. Endocrinology 133, 1830–1842 (1993)PubMedGoogle Scholar
  10. 10.
    R. Arumugam, D. Fleenor, D. Lu, M. Freemark, Differential and complementary effects of glucose and prolactin on islet DNA synthesis and gene expression. Endocrinology 152, 856–868 (2011)PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    R. Arumugam, E. Horowitz, D. Lu et al., The interplay of prolactin and the glucocorticoids in the regulation of beta-cell gene expression, fatty acid oxidation, and glucose-stimulated insulin secretion: implications for carbohydrate metabolism in pregnancy. Endocrinology 149, 5401–5414 (2008)PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    R. Arumugam, E. Horowitz, R.C. Noland, D. Lu, D. Fleenor, M. Freemark, Regulation of islet beta-cell pyruvate metabolism: interactions of prolactin, glucose, and dexamethasone. Endocrinology 151, 3074–3083 (2010)PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    T.C. Brelje, D.W. Scharp, P.E. Lacy et al., Effect of homologous placental lactogens, prolactins, and growth hormones on islet B-cell division and insulin secretion in rat, mouse, and human islets: implication for placental lactogen regulation of islet function during pregnancy. Endocrinology 132, 879–887 (1993)PubMedGoogle Scholar
  14. 14.
    M. Freemark, Ontogenesis of prolactin receptors in the human fetus: roles in fetal development. Biochem. Soc. Trans. 29, 38–41 (2001)PubMedCrossRefGoogle Scholar
  15. 15.
    L. Labriola, W.R. Montor, K. Krogh et al., Beneficial effects of prolactin and laminin on human pancreatic islet-cell cultures. Mol. Cell. Endocrinol. 263, 120–133 (2007)PubMedCrossRefGoogle Scholar
  16. 16.
    A. Petryk, D. Fleenor, P. Driscoll, M. Freemark, Prolactin induction of insulin gene expression: the roles of glucose and glucose transporter-2. J. Endocrinol. 164, 277–286 (2000)PubMedCrossRefGoogle Scholar
  17. 17.
    T. Yamamoto, C. Ricordi, A. Mita et al., beta-Cell specific cytoprotection by prolactin on human islets. Transpl. Proc. 40, 382–383 (2008)CrossRefGoogle Scholar
  18. 18.
    D. Fleenor, A. Petryk, P. Driscoll, M. Freemark, Constitutive expression of placental lactogen in pancreatic beta cells: effects on cell morphology, growth, and gene expression. Pediatr. Res. 47, 136–142 (2000)PubMedCrossRefGoogle Scholar
  19. 19.
    R.C. Vasavada, A. Garcia-Ocana, W.S. Zawalich et al., Targeted expression of placental lactogen in the beta cells of transgenic mice results in beta cell proliferation, islet mass augmentation, and hypoglycemia. J. Biol. Chem. 275, 15399–15406 (2000)PubMedCrossRefGoogle Scholar
  20. 20.
    M. Freemark, I. Avril, D. Fleenor et al., Targeted deletion of the PRL receptor: effects on islet development, insulin production, and glucose tolerance. Endocrinology 143, 1378–1385 (2002)PubMedCrossRefGoogle Scholar
  21. 21.
    C. Huang, F. Snider, J.C. Cross, Prolactin receptor is required for normal glucose homeostasis and modulation of beta-cell mass during pregnancy. Endocrinology 150, 1618–1626 (2009)PubMedCrossRefGoogle Scholar
  22. 22.
    S.K. Karnik, H. Chen, G.W. McLean et al., Menin controls growth of pancreatic beta-cells in pregnant mice and promotes gestational diabetes mellitus. Science 318, 806–809 (2007)PubMedCrossRefGoogle Scholar
  23. 23.
    H. Kim, Y. Toyofuku, F.C. Lynn, Serotonin regulates pancreatic beta cell mass during pregnancy. Nat. Med. 16, 804–808 (2010)PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    A. Schraenen, K. Lemaire, G. de Faudeur et al., Placental lactogens induce serotonin biosynthesis in a subset of mouse beta cells during pregnancy. Diabetologia 53, 2589–2599 (2010)PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    T.C. Becker, R.J. Noel, W.S. Coats et al., Use of recombinant adenovirus for metabolic engineering of mammalian cells. Methods Cell Biol. 43, 161–189 (1994)PubMedCrossRefGoogle Scholar
  26. 26.
    H.E. Hohmeier, H. Mulder, G. Chen, R. Henkel-Rieger, M. Prentki, C.B. Newgard, Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion. Diabetes 49, 424–430 (2000)PubMedCrossRefGoogle Scholar
  27. 27.
    M.D. Holland, K.L. Hossner, S.E. Williams, C.R. Wallace, G.D. Niswender, K.G. Odde, Serum concentrations of insulin-like growth factors and placental lactogen during gestation in cattle. I. Fetal profiles. Domest. Anim. Endocrinol. 14, 231–239 (1997)PubMedCrossRefGoogle Scholar
  28. 28.
    B.N. Friedrichsen, H.E. Richter, J.A. Hansen et al., Signal transducer and activator of transcription 5 activation is sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic beta-cells. Mol. Endocrinol. 17, 945–958 (2003)PubMedCrossRefGoogle Scholar
  29. 29.
    S. Georgia, A. Bhushan, Beta cell replication is the primary mechanism for maintaining postnatal beta cell mass. J. Clin. Invest. 114, 963–968 (2004)PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    J.A. Kushner, M.A. Ciemerych, E. Sicinska et al., Cyclins D2 and D1 are essential for postnatal pancreatic beta-cell growth. Mol. Cell. Biol. 25, 3752–3762 (2005)PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    S. Rieck, P. White, J. Schug et al., The transcriptional response of the islet to pregnancy in mice. Mol. Endocrinol. 23, 1702–1712 (2009)PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    H. Zhang, J. Zhang, C.F. Pope et al., Gestational diabetes mellitus resulting from impaired beta-cell compensation in the absence of FoxM1, a novel downstream effector of placental lactogen. Diabetes 59, 143–152 (2010)PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    N. Kubota, K. Tobe, Y. Terauchi et al., Disruption of insulin receptor substrate 2 causes type 2 diabetes because of liver insulin resistance and lack of compensatory beta-cell hyperplasia. Diabetes 49, 1880–1889 (2000)PubMedCrossRefGoogle Scholar
  34. 34.
    Y. Fujinaka, K. Takane, H. Yamashita, R.C. Vasavada, Lactogens promote beta cell survival through JAK2/STAT5 activation and Bcl-XL upregulation. J. Biol. Chem. 282, 30707–30717 (2007)PubMedCrossRefGoogle Scholar
  35. 35.
    N.G. Kondegowda, A. Mozar, C. Chin, A. Otero, A. Garcia-Ocana, R.C. Vasavada, Lactogens protect rodent and human beta cells against glucolipotoxicity-induced cell death through Janus kinase-2 (JAK2)/signal transducer and activator of transcription-5 (STAT5) signalling. Diabetologia 55, 1721–1732 (2012)PubMedCrossRefGoogle Scholar
  36. 36.
    V. Chesnokova, C. Wong, S. Zonis et al., Diminished pancreatic beta-cell mass in securin-null mice is caused by beta-cell apoptosis and senescence. Endocrinology 150, 2603–2610 (2009)PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    R. Yu, M. Cruz-Soto, S. Li Calzi, H. Hui, S. Melmed, Murine pituitary tumor-transforming gene functions as a securin protein in insulin-secreting cells. J. Endocrinol. 191, 45–53 (2006)PubMedCrossRefGoogle Scholar
  38. 38.
    T.H. Tran, F.E. Utama, J. Lin et al., Prolactin inhibits BCL6 expression in breast cancer through a Stat5a-dependent mechanism. Cancer Res. 70, 1711–1721 (2010)PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    S.R. Walker, E.A. Nelson, D.A. Frank, STAT5 represses BCL6 expression by binding to a regulatory region frequently mutated in lymphomas. Oncogene 26, 224–233 (2007)PubMedCrossRefGoogle Scholar
  40. 40.
    H. Xiong, W.Y. Su, Q.C. Liang et al., Inhibition of STAT5 induces G1 cell cycle arrest and reduces tumor cell invasion in human colorectal cancer cells. Lab. Invest. 89, 717–725 (2009)PubMedCrossRefGoogle Scholar
  41. 41.
    R.D. Meyer, E.V. Laz, T. Su, D.J. Waxman, Male-specific hepatic Bcl6: growth hormone-induced block of transcription elongation in females and binding to target genes inversely coordinated with STAT5. Mol. Endocrinol. 23, 1914–1926 (2009)PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    S.C. Martinez, C. Cras-Meneur, E. Bernal-Mizrachi, M.A. Permutt, Glucose regulates FoxO1 through insulin receptor signaling in the pancreatic islet beta-cell. Diabetes 55, 1581–1591 (2006)PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Department of Pediatric Endocrinology & DiabetesDuke University Medical CenterDurhamUSA
  2. 2.Department of Cell BiologyDuke University Medical CenterDurhamUSA

Personalised recommendations