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Arthur Gutierrez-Hartmann, Professor

M.D. (1975), University of Texas Southwestern Medical School
 

 

 

Contact Info:

Molecular Biology
University of Colorado

Arthur Gutierrez-Hartmann, Ph.D.  Research One South
(RC1-South), Room 7108 Gutierrez-Hartmann@ucdenver.edu Phone: 303-724-3921

The main focus of my laboratory is to determine the role of Ras/MAPK signaling and Ets transcription factors in epithelial cell development and tumorigenesis, with a focus on pituitary and mammary model systems.

With regards to the pituitary project, we study how the combinatorial action of Ets factors, Ets-1 and GABP, acting in concert with the POU-homeodomain transcription factor, Pit-1, serve to regulate the lactotrope-specific basal and Ras-regulated expression of the prolactin gene. Utilizing biochemical, structural, molecular and transgenic approaches, we have discovered that Ets factors play a critical role in specifying lactotrope cell identity in pituitary ontogeny. Using transgenic mice, we show that targeting a dominant-negative Ets transgene to the pituitary lactotrope lineage abrogates pituitary lactotrope development. We are also studying the biological role of Pit-1ß, an alternative-splice form of Pit-1, which appears to act as a negative regulator of Pit-1 functions. We also seek to elucidate the molecular mechanisms by which lactotrope adenomas arise and why they rarely progress to frank carcinoma. To this end, we have generated GH4 pituitary cell lines stably expressing a doxycycline-regulated HA-V12Ras, to determine whether persistent vs short-term MAPK activation promotes cell proliferation, differentiation or apoptosis. Finally, we also plan to address the role of adult pituitary progenitor/stem cells in facultative lactotrope expansion during pregnancy and in tumorigenesis.

With regards to our breast cancer project, we study the role of the epithelial-specific Ets transcription factor, Ese-1, in human mammary epithelial cell tumorigenesis. Ese-1 is over-expressed in many breast cancers and, thus, it is particularly relevant in human disease. We have shown that Ese-1 confers a transformed phenotype to immortalized, but nontransformed MCF-12A human mammary epithelial cells via a novel cytoplasmic mechanism. We also demonstrated that exclusive cytoplasmic targeting of a unique, 40-AA serine- and aspartic rich (SAR) subdomain is both necessary and sufficient for the transformation response. Finally, using anti-Ese-1 monoclonal antibodies that we developed, we found Ese-1 to be expressed in the nucleus in established breast cancer cell lines, and we demonstrated that Ese-1 is required to maintain the transformed state by using shRNA knockdown of endogenous Ese-1 in these cells. Taken together, these studies reveal that Ese-1, as a sole agent, acts via a cytoplasmic mechanism to initiate transformation, but once mammary cells are fully transformed, via the action of several mechanisms, then nuclear Ese-1 is required to maintain the malignant phenotype.

We are making a significant commitment to inducible-methods and transgenic approaches to decipher the role of Ras/MAPK signaling, and specific POU-homeodomain and Ets transcription factors in mediating the ontogeny, maintenance and tumorigenesis of epithelial cells in the pituitary and mammary systems.

                        Figure 4. Mapping of chemical shift perturbations to the Pithd structure.

Fig4


Surface density representations (inlays: ribbon representation in the same orientation) of the crystal structure of the Pithd bound to DNA. These fragments were taken from the crystal structure as resolved by Jacobson et al (1997).
  1. The results from figure 2C have been indicated by color coding. Chemical shift changes > 25 Hz are colored red, changes between 25 and 15 Hz dark red and between 15 and 10 pink. Unaffected residues are colored yellow. In green are the solvent exposed residues of the first helix, which showed a periodicity of three residues in chemical shift perturbation. The amide proton of W261, which is deeply buried, is colored in blue.
  2. The hydrophobic pocket around W261 formed by V257, V258, I222 and W261 itself is colored in beige. Overlaying the pocket is K226, colored in blue. The DNA has been deleted for clarity.
  3. Model for association of Ets-1 to the Pithd. K226 is shown in blue, the hydrophobic pocket in beige and T220 in green. Since the crystal structure does not extend beyond residue K273, an extended tail was added to represent the C-terminus of the Pithd. Ets-1 could use L288 for initial binding and subsequently, depending on the post-translational state of the Pithd, dock in the hydrophobic pocket next to W261. Additional stabilizing contacts may be made at the C-terminus of the DNA recognition helix (helix 3). These pictures were generated using MSI's Weblab viewer Lite version (www.msi.com).

 

Wasylyk B, Hagman, J, Gutierrez-Hartmann, A. ETS transcription factors: Nuclear effectors of the Ras/MAPK signaling pathway. Trends in Bioch. Sci. 23: 213-216, 1998.

Diamond SE, Chiono M, Gutierrez-Hartmann A. Reconstitution of the protein kinase A response of the rat prolactin promoter: Differential effects of distinct Pit-1 isoforms and functional interaction with Oct-1. Molecular Endo 13:228-238, 1998.

Farrow KN, Gutierrez-Hartmann A. Transforming Growth Factor-ß1 inhibits rat prolactin promoter activity in GH4 neuroendocrine cells. DNA & Cell Biol 18:863-873, 1999.

Bradford AP, Brodsky KS, Diamond SE, Kuhn LC, Liu Y, Gutierrez-Hartmann A. The Pit-1 homeodomain and ß-domain interact with Ets-1 and modulate synergistic activation of the rat prolactin promoter. J. Biol. Chem. 275: 3100-3106, 2000.

Diamond SE, Gutierrez-Hartmann A. The Pit-1ß-domain dictates active repression and alteration of histone acetylation of the proximal rat prolactin promoter. J. Biol. Chem. 275: 30977-30986, 2000.

Schweppe RE, Gutierrez-Hartmann A. Pituitary Ets-1 and GABP bind to the growth factor regulatory sites of the rat prolactin promoter. Nucl. Acids Res. 29:1251-1260, 2001.

Dunkelburg JA, Gutierrez-Hartmann A.LZ-FYVE: A novel developmental stage-specific leucine zipper, FYVE-finger protein. DNA & Cell Biology 20:403-412, 2001.

Augustijn KD*, Duvall DL*, Wechselberger R, Kaptein R, Gutierrez-Hartmann A, van der Vliet, PC. Structural characterization of the Pit-1/Ets-1 interaction: Pit-1 phosphorylation regulates Pit-1/Ets-1 binding. Proc. Natl. Acad. Sci. USA 99:12657-12662, 2002. (* dual first authors).

Pickett CA, Manning N, Akita Y, Gutierrez-Hartmann A. Role of specific protein kinase C isozymes in mediating epidermal growth factor, thyrotropin-releasing hormone, and phorbol ester regulation of the rat prolactin promoter in GH4/GH4C1 pituitary cells. Mol Endocrinol. 16:2840-2852, 2002.

Eckel KL, Diamond SE, Tentler JJ, Cappetta GJ, Gutierrez-Hartmann A. The epithelial-specific ETS transcription factor ESX/ESE-1/Elf-3 modulates breast cancer-associated gene expression. DNA & Cell Biol. 22:79-94, 2003.

Tentler JJ, AP Bradford, RE Schweppe, Gutierrez-Hartmann A. Selective repression of prolactin gene transcription by stable expression of dominant-negative Ets in GH4 rat pituitary cells. Endocrine, in press, 2003.

Schweppe RE, Melton AA, Eads M, Aveline L, Resing, K, Ahn NG, Gutierrez-Hartmann A. Purification and mass spectrometric identification of GABP as the functional pituitary Ets factor binding to the basal transcription element of the prolactin promoter. J. Biol. Chem., in press, 2003.

Schedin PJ, Eckel KL, McDaniel SM, Prescott JD, Brodsky KS, Tentler JJ, Gutierrez-Hartmann A. ESX induces transformation and functional epithelial to mesenchymal transition in MCF-12A mammary epithelial cells. Submitted.

Duval DL, Jean A, Gutierrez-Hartmann A. Ras signaling and transcriptional synergy at a flexible Ets-1/Pit-1 composite element is defined by the assembly of selective activation domains. Submitted.

Bennett, W.F., Gutierrez-Hartmann, A. and Butow, R.A. The role of mitochondria-bound 80S ribosomes in mitochondrial biogenesis. IN: Interdisciplinary Conference on the Genetics and Biogenesis of Mitochondria and Chloroplasts. Amsterdam: North Holland Press, 1977.

Gutierrez-Hartmann, A. Basics of transcription control. IN: Introduction to Endocrine Investigation 1988: Techniques and Concepts. Serono Symposia, USA Press, 1988.

Gutierrez-Hartmann, A. Pituitary-specific expression of the rat prolactin gene. IN: Molecular and Clinical Advances in Pituitary Disorders. S. Melmed and R.J. Robbins, Eds., Blackwell Scientific Publication: Cambridge, MA, 1991.

Jackson, S.M., Barnhart, K.M., Mellon, P.L., Gutierrez-Hartmann, A. and Hoeffler, J.P. The role of helix-loop-helix proteins in gonadotropin gene expression. IN: Glycoprotein Hormones: Structure, Function and Clinical Implications. JW Lustbader, JD Puett, and R Ruddon, Eds. Plenum Publishing Corp., NY, 1993.

Pickett, C.A. and Gutierrez-Hartmann, A. Molecular and cellular ontogeny of distinct pituitary cell types. IN: Pituitary Disease: Diagnosis and Treatment. ME Wierman, Ed. Humana Press, Totowa, NY, In Press, 1996.