Prostate-specific Klf6 Inactivation Impairs Anterior Prostate Branching Morphogenesis through Increased Activation of the Shh Pathway
2009; Elsevier BV; Volume: 284; Issue: 31 Linguagem: Inglês
10.1074/jbc.m109.001776
ISSN1083-351X
AutoresChing Ching Leow, Bu-Er Wang, Jed Ross, Sara M. Chan, Jiping Zha, Richard A.D. Carano, Gretchen Frantz, Michael M. Shen, Frédéric J. de Sauvage, Wei‐Qiang Gao,
Tópico(s)Epigenetics and DNA Methylation
ResumoKrüppel-like factor 6 (Klf6) belongs to a family of zinc finger transcription factors known to play a role in development and tumor suppression. Although Klf6 is highly mutated in prostate cancer, its function in prostate development is unknown. We have generated a prostate-specific Klf6-deficient mouse model and report here a novel role for Klf6 in the regulation of prostate branching morphogenesis. Importantly, our study reveals a novel relationship between Klf6 and the Shh pathway. Klf6-deficiency leads to elevated levels of hedgehog pathway components (Shh, Ptc, and Gli) and loss of their localized expression, which in turn causes impaired lateral branching. Krüppel-like factor 6 (Klf6) belongs to a family of zinc finger transcription factors known to play a role in development and tumor suppression. Although Klf6 is highly mutated in prostate cancer, its function in prostate development is unknown. We have generated a prostate-specific Klf6-deficient mouse model and report here a novel role for Klf6 in the regulation of prostate branching morphogenesis. Importantly, our study reveals a novel relationship between Klf6 and the Shh pathway. Klf6-deficiency leads to elevated levels of hedgehog pathway components (Shh, Ptc, and Gli) and loss of their localized expression, which in turn causes impaired lateral branching. Klf6 belongs to the family of Krüppel-like zinc finger transcription factors that regulate cell proliferation and differentiation (1Bieker J.J. J. Biol. Chem. 2001; 276: 34355-34358Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). All members of the Klf gene family contain a highly conserved zinc finger DNA binding domain at their C terminus and an activation domain at its N terminus, distinct to each Klf gene and accounting for their wide-ranging biological capabilities (2Huber T.L. Perkins A.C. Deconinck A.E. Chan F.Y. Mead P.E. Zon L.I. Curr. Biol. 2001; 11: 1456-1461Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 3Laub F. Aldabe R. Friedrich Jr., V. Ohnishi S. Yoshida T. Ramirez F. Dev. Biol. 2001; 233: 305-318Crossref PubMed Scopus (78) Google Scholar, 4Oates A.C. Pratt S.J. Vail B. Yan Y.I. Ho R.K. Johnson S.L. Postlethwait J.H. Zon L.I. Blood. 2001; 98: 1792-1801Crossref PubMed Scopus (92) Google Scholar). Similar to other members of the Klf family (5Zhao W. Hisamuddin I.M. Nandan M.O. Babbin B.A. Lamb N.E. Yang V.W. Oncogene. 2004; 23: 395-402Crossref PubMed Scopus (263) Google Scholar, 6Chen C. Bhalala H.V. Qiao H. Dong J.T. Oncogene. 2002; 21: 6567-6572Crossref PubMed Scopus (127) Google Scholar), Klf6 is reported to act as a tumor suppressor (7Narla G. Heath K.E. Reeves H.L. Li D. Giono L.E. Kimmelman A.C. Glucksman M.J. Narla J. Eng F.J. Chan A.M. Ferrari A.C. Martignetti J.A. Friedman S.L. Science. 2001; 294: 2563-2566Crossref PubMed Scopus (377) Google Scholar, 8Reeves H.L. Narla G. Ogunbiyi O. Haq A.I. Katz A. Benzeno S. Hod E. Harpaz N. Goldberg S. Tal-Kremer S. Eng F.J. Arthur M.J. Martignetti J.A. Friedman S.L. Gastroenterology. 2004; 126: 1090-1103Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 9Kimmelman A.C. Qiao R.F. Narla G. Banno A. Lau N. Bos P.D. Nuñez Rodriguez N. Liang B.C. Guha A. Martignetti J.A. Friedman S.L. Chan A.M. Oncogene. 2004; 23: 5077-5083Crossref PubMed Scopus (67) Google Scholar, 10Ito G. Uchiyama M. Kondo M. Mori S. Usami N. Maeda O. Kawabe T. Hasegawa Y. Shimokata K. Sekido Y. Cancer Res. 2004; 64: 3838-3843Crossref PubMed Scopus (131) Google Scholar). Klf6 has been reported to be mutated in a large percentage of human prostate tumors (7Narla G. Heath K.E. Reeves H.L. Li D. Giono L.E. Kimmelman A.C. Glucksman M.J. Narla J. Eng F.J. Chan A.M. Ferrari A.C. Martignetti J.A. Friedman S.L. Science. 2001; 294: 2563-2566Crossref PubMed Scopus (377) Google Scholar), but its function during normal development has not been elucidated.Prostate development, specifically epithelial branching morphogenesis, is a well studied process. Outgrowth and branching of the prostate epithelial buds into the enveloping mesenchyme occur during the first 3 weeks postnatally (11Sugimura Y. Cunha G.R. Donjacour A.A. Biol. Reprod. 1986; 34: 961-971Crossref PubMed Scopus (311) Google Scholar). The fully developed prostate in an adult mouse is composed of three different paired lobes, referred as the anterior, ventral, and dorsal-lateral lobes. In addition, the number of main ducts and complexity of ductal branching vary among the three lobes. A number of growth factors/pathways have been implicated in regulating prostatic epithelial proliferation and differentiation, including hedgehog (Hh), 4The abbreviations used are: HhhedgehogBMPbone morphogenic proteinFGFfibroblastic growth factorShhsonic hedgehogMicro-CTmicro-computed tomographySMAsmooth muscle actinH&Ehematoxylin and eosinTCRDT-cell receptor delta chain. 4The abbreviations used are: HhhedgehogBMPbone morphogenic proteinFGFfibroblastic growth factorShhsonic hedgehogMicro-CTmicro-computed tomographySMAsmooth muscle actinH&Ehematoxylin and eosinTCRDT-cell receptor delta chain. bone morphogenic proteins (BMPs), fibroblastic growth factors (FGFs), and Notch and Wnt pathways (12Freestone S.H. Marker P. Grace O.C. Tomlinson D.C. Cunha G.R. Harnden P. Thomson A.A. Dev. Biol. 2003; 264: 352-362Crossref PubMed Scopus (128) Google Scholar, 13Wang B.E. Shou J. Ross S. Koeppen H. De Sauvage F.J. Gao W.Q. J. Biol. Chem. 2003; 278: 18506-18513Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 14Berman D.M. Desai N. Wang X. Karhadkar S.S. Reynon M. Abate-Shen C. Beachy P.A. Shen M.M. Dev. Biol. 2004; 267: 387-398Crossref PubMed Scopus (111) Google Scholar, 15Lamm M.L. Catbagan W.S. Laciak R.J. Barnett D.H. Hebner C.M. Gaffield W. Walterhouse D. Iannaccone P. Bushman W. Dev. Biol. 2002; 249: 349-366Crossref PubMed Scopus (137) Google Scholar, 16Thomson A.A. Cunha G.R. Development. 1999; 126: 3693-3701PubMed Google Scholar, 17Lamm M.L. Podlasek C.A. Barnett D.H. Lee J. Clemens J.Q. Hebner C.M. Bushman W. Dev. Biol. 2001; 232: 301-314Crossref PubMed Scopus (124) Google Scholar, 18Wang X.D. Leow C.C. Zha J. Tang Z. Modrusan Z. Radtke F. Aguet M. de Sauvage F.J. Gao W.Q. Dev. Biol. 2006; 290: 66-80Crossref PubMed Scopus (122) Google Scholar, 19Wang B.E. Wang X.D. Ernst J.A. Polakis P. Gao W.Q. PLoS ONE. 2008; 3: e2186Crossref PubMed Scopus (50) Google Scholar). For example, work done in our laboratory and others showed that sonic hedgehog (Shh) produced in the prostatic epithelium is a negative regulator of prostatic branching morphogenesis, mediated indirectly by periepithelial mesenchymal cells (12Freestone S.H. Marker P. Grace O.C. Tomlinson D.C. Cunha G.R. Harnden P. Thomson A.A. Dev. Biol. 2003; 264: 352-362Crossref PubMed Scopus (128) Google Scholar, 13Wang B.E. Shou J. Ross S. Koeppen H. De Sauvage F.J. Gao W.Q. J. Biol. Chem. 2003; 278: 18506-18513Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 14Berman D.M. Desai N. Wang X. Karhadkar S.S. Reynon M. Abate-Shen C. Beachy P.A. Shen M.M. Dev. Biol. 2004; 267: 387-398Crossref PubMed Scopus (111) Google Scholar, 15Lamm M.L. Catbagan W.S. Laciak R.J. Barnett D.H. Hebner C.M. Gaffield W. Walterhouse D. Iannaccone P. Bushman W. Dev. Biol. 2002; 249: 349-366Crossref PubMed Scopus (137) Google Scholar). In addition, while mesenchymal FGF10 stimulates prostatic epithelial growth (16Thomson A.A. Cunha G.R. Development. 1999; 126: 3693-3701PubMed Google Scholar), BMP4 is a mesenchymal factor that inhibits prostatic ductal budding and branching morphogenesis (17Lamm M.L. Podlasek C.A. Barnett D.H. Lee J. Clemens J.Q. Hebner C.M. Bushman W. Dev. Biol. 2001; 232: 301-314Crossref PubMed Scopus (124) Google Scholar).All Klf knockouts generated thus far are embryonic lethal (20Kuo C.T. Veselits M.L. Barton K.P. Lu M.M. Clendenin C. Leiden J.M. Genes Dev. 1997; 11: 2996-3006Crossref PubMed Scopus (304) Google Scholar, 21Nuez B. Michalovich D. Bygrave A. Ploemacher R. Grosveld F. Nature. 1995; 375: 316-318Crossref PubMed Scopus (476) Google Scholar, 22Segre J.A. Bauer C. Fuchs E. Nat. Genet. 1999; 22: 356-360Crossref PubMed Scopus (630) Google Scholar), including the Klf6 knock-out mice (23Matsumoto N. Kubo A. Liu H. Akita K. Laub F. Ramirez F. Keller G. Friedman S.L. Blood. 2006; 107: 1357-1365Crossref PubMed Scopus (101) Google Scholar). Because prostate development begins in late gestation and continues postnatally, we overcame embryonic lethality to study the role of Klf6 in prostate development and tumorigenesis by generating a prostate-specific deletion of the Klf6 gene using a Cre-lox recombination approach. We report here a novel role for Klf6 in the regulation of Shh-mediated epithelial branching morphogenesis in the anterior prostate.DISCUSSIONThe Klf gene family is known to be crucial for development in vertebrates (4Oates A.C. Pratt S.J. Vail B. Yan Y.I. Ho R.K. Johnson S.L. Postlethwait J.H. Zon L.I. Blood. 2001; 98: 1792-1801Crossref PubMed Scopus (92) Google Scholar). They are expressed in a large number of tissues (40Turner J. Crossley M. Trends Biochem. Sci. 1999; 24: 236-240Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar) and play an important role in the control of hematopoietic cell differentiation (4Oates A.C. Pratt S.J. Vail B. Yan Y.I. Ho R.K. Johnson S.L. Postlethwait J.H. Zon L.I. Blood. 2001; 98: 1792-1801Crossref PubMed Scopus (92) Google Scholar), erythroid cell maturation (21Nuez B. Michalovich D. Bygrave A. Ploemacher R. Grosveld F. Nature. 1995; 375: 316-318Crossref PubMed Scopus (476) Google Scholar, 41Perkins A.C. Gaensler K.M. Orkin S.H. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 12267-12271Crossref PubMed Scopus (127) Google Scholar), T-cell activation (42Kuo C.T. Veselits M.L. Leiden J.M. Science. 1997; 277: 1986-1990Crossref PubMed Scopus (342) Google Scholar), blood vessel stability (20Kuo C.T. Veselits M.L. Barton K.P. Lu M.M. Clendenin C. Leiden J.M. Genes Dev. 1997; 11: 2996-3006Crossref PubMed Scopus (304) Google Scholar), and skin permeability (22Segre J.A. Bauer C. Fuchs E. Nat. Genet. 1999; 22: 356-360Crossref PubMed Scopus (630) Google Scholar). Using the prostate as a model, we describe here for the first time a role for Klf6 in prostate branching morphogenesis. Consistent with this notion, Klf14, another Klf family member, has been shown to be critical for alveolarization in the developing lung. Deletion of Klf14 leads to thinner lung alveolar walls and poor outgrowth of secondary septa, which results in defective blood-air exchange and respiratory failures in newborn mice (43Hertveldt V. Louryan S. van Reeth T. Drèze P. van Vooren P. Szpirer J. Szpirer C. Dev. Dyn. 2008; 237: 883-892Crossref PubMed Scopus (26) Google Scholar). In the kidney, Klf6 is known to be expressed in the Wolffian duct, uteric bud, collecting ducts, and mesangium (44Fischer E.A. Verpont M.C. Garrett-Sinha L.A. Ronco P.M. Rossert J.A. J. Am Soc. Nephrol. 2001; 12: 726-735Crossref PubMed Google Scholar), an expression profile that would also be consistent with a role in branching morphogenesis.Because systemic loss of Klf6 is embryonic lethal (23Matsumoto N. Kubo A. Liu H. Akita K. Laub F. Ramirez F. Keller G. Friedman S.L. Blood. 2006; 107: 1357-1365Crossref PubMed Scopus (101) Google Scholar), we generated mice deficient for Klf6 specifically in the prostate. The resulting lateral branching defect in Klf6-deficient prostate was confirmed by light microscopy, H&E staining, and micro-CT imaging. Using a novel ex vivo ultrasound imaging modality, we were able to demonstrate that Klf6 plays a role in modulating the epithelial infolding and tufting, which results in decreased surface area of functional epithelium. The most prominent branching defects are seen in the anterior prostate, also known as a coagulating gland, which correlates with the highest level of Klf6 depletion (Fig. 1E). Despite the reduction in epithelial infolding and tufting whether or not prostatic function has been impaired in these mice remains to be determined.The augmented branching defect in the anterior prostate is noteworthy, because the branching patterns in the anterior prostate are distinct from the dorsolateral prostate and ventral prostate. In addition, the embryologic origin of the anterior prostate is also different from the other prostate lobes (45Cunha G.R. Lung B. Accesory Glands of Male Reproductive Tract. 6. Ann Arbor Science, Ann Arbor, MI1979: 1-28Google Scholar, 46Cunha G.R. Alarid E.T. Turner T. Donjacour A.A. Boutin E.L. Foster B.A. J. Androl. 1992; 13: 465-475PubMed Google Scholar). Although branching patterns of the ventral prostate and dorsolateral prostates are believed to arise from the urogenital sinus epithelium invaginating into urogenital sinus mesenchyme, the anterior prostate is thought to be derived from urogenital sinus epithelium at the most dorsal aspect of the urogenital sinus invaginating into the mesenchyme of the seminal vesicle, which itself is derived from the mesodermal Wolffian duct. Hence, the resulting branched structure in the anterior prostate has unique patterns distinct from the other prostate lobes and occurs as a result of lateral branch events and not as a result of bifurcations at the elongating tips.Consistent with previous reports from our group and others on the role of Shh signaling in prostate branching (12Freestone S.H. Marker P. Grace O.C. Tomlinson D.C. Cunha G.R. Harnden P. Thomson A.A. Dev. Biol. 2003; 264: 352-362Crossref PubMed Scopus (128) Google Scholar, 13Wang B.E. Shou J. Ross S. Koeppen H. De Sauvage F.J. Gao W.Q. J. Biol. Chem. 2003; 278: 18506-18513Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 14Berman D.M. Desai N. Wang X. Karhadkar S.S. Reynon M. Abate-Shen C. Beachy P.A. Shen M.M. Dev. Biol. 2004; 267: 387-398Crossref PubMed Scopus (111) Google Scholar, 15Lamm M.L. Catbagan W.S. Laciak R.J. Barnett D.H. Hebner C.M. Gaffield W. Walterhouse D. Iannaccone P. Bushman W. Dev. Biol. 2002; 249: 349-366Crossref PubMed Scopus (137) Google Scholar, 37Pu Y. Huang L. Prins G.S. Dev. Biol. 2004; 273: 257-275Crossref PubMed Scopus (89) Google Scholar), both Ptc-lacZ reporter mice crossed to Klf6f/fNkx3.1Cre/+ mice and in situ hybridization data in the present study indicate that impaired lateral branching in the Klf6-deficient prostate correlate with an up-regulation and loss of spatial localization of Shh, Ptc, and Gli1. Further support for the involvement of focal Hh signaling in prostate epithelial branching comes from a study by Bushman and co-workers (15Lamm M.L. Catbagan W.S. Laciak R.J. Barnett D.H. Hebner C.M. Gaffield W. Walterhouse D. Iannaccone P. Bushman W. Dev. Biol. 2002; 249: 349-366Crossref PubMed Scopus (137) Google Scholar), showing a close association between prostate ductal bud formation and localized expression of Shh at the growing tips of elongating prostate ducts. In addition, Shh regulates prostate branching morphogenesis in concert with FGF10 and BMP4 (37Pu Y. Huang L. Prins G.S. Dev. Biol. 2004; 273: 257-275Crossref PubMed Scopus (89) Google Scholar). Shh secreted from the epithelial ductal tips can down-regulate FGF10 that stimulates epithelial cell growth (16Thomson A.A. Cunha G.R. Development. 1999; 126: 3693-3701PubMed Google Scholar) and/or up-regulate BMP4 that inhibits epithelial cell growth (17Lamm M.L. Podlasek C.A. Barnett D.H. Lee J. Clemens J.Q. Hebner C.M. Bushman W. Dev. Biol. 2001; 232: 301-314Crossref PubMed Scopus (124) Google Scholar) in the mesenchyme, which results in epithelial ductal branching (37Pu Y. Huang L. Prins G.S. Dev. Biol. 2004; 273: 257-275Crossref PubMed Scopus (89) Google Scholar). In the present study, we show that loss of Klf6 leads to dispersed, rather than focal, Shh expression throughout the epithelial ducts and corresponding dispersed Ptc and Gli1 expression in the adjacent mesenchyme (Fig. 5). This broader domain of Hh pathway activity leads to an up-regulation of BMP4 expression, resulting in reduced ductal branching. As proposed by Pu and coworkers, the interplay and localized expression pattern of Shh and BMP4 are key to conveying critical branching information (37Pu Y. Huang L. Prins G.S. Dev. Biol. 2004; 273: 257-275Crossref PubMed Scopus (89) Google Scholar).Klf6 is reported to be a tumor suppressor that is inactivated in a large subset of prostate cancer (47Narla G. Friedman S.L. Martignetti J.A. Am. J. Pathol. 2003; 162: 1047-1052Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). However, our detailed histological characterization of Klf6f/fNkx3.1+/+ mice as old as 2 years failed to reveal any prostate tumors. 7C. C. Leow, B. Wang, J. Ross, S. M. Chan, J. Zha, R. A. D. Carano, G. Frantz, M. M. Shen, F. J. de Sauvage, and W.-Q. Gao, unpublished observations. The lack of tumor phenotype in the Klf6 mutant mice could be due to compensations/redundancy with other Klf family members. It is also possible that loss of Klf6 might not be sufficient to initiate prostate tumorigenesis in the mouse and that loss/mutation of additional tumor suppressors might be required. Klf6 belongs to the family of Krüppel-like zinc finger transcription factors that regulate cell proliferation and differentiation (1Bieker J.J. J. Biol. Chem. 2001; 276: 34355-34358Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). All members of the Klf gene family contain a highly conserved zinc finger DNA binding domain at their C terminus and an activation domain at its N terminus, distinct to each Klf gene and accounting for their wide-ranging biological capabilities (2Huber T.L. Perkins A.C. Deconinck A.E. Chan F.Y. Mead P.E. Zon L.I. Curr. Biol. 2001; 11: 1456-1461Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 3Laub F. Aldabe R. Friedrich Jr., V. Ohnishi S. Yoshida T. Ramirez F. Dev. Biol. 2001; 233: 305-318Crossref PubMed Scopus (78) Google Scholar, 4Oates A.C. Pratt S.J. Vail B. Yan Y.I. Ho R.K. Johnson S.L. Postlethwait J.H. Zon L.I. Blood. 2001; 98: 1792-1801Crossref PubMed Scopus (92) Google Scholar). Similar to other members of the Klf family (5Zhao W. Hisamuddin I.M. Nandan M.O. Babbin B.A. Lamb N.E. Yang V.W. Oncogene. 2004; 23: 395-402Crossref PubMed Scopus (263) Google Scholar, 6Chen C. Bhalala H.V. Qiao H. Dong J.T. Oncogene. 2002; 21: 6567-6572Crossref PubMed Scopus (127) Google Scholar), Klf6 is reported to act as a tumor suppressor (7Narla G. Heath K.E. Reeves H.L. Li D. Giono L.E. Kimmelman A.C. Glucksman M.J. Narla J. Eng F.J. Chan A.M. Ferrari A.C. Martignetti J.A. Friedman S.L. Science. 2001; 294: 2563-2566Crossref PubMed Scopus (377) Google Scholar, 8Reeves H.L. Narla G. Ogunbiyi O. Haq A.I. Katz A. Benzeno S. Hod E. Harpaz N. Goldberg S. Tal-Kremer S. Eng F.J. Arthur M.J. Martignetti J.A. Friedman S.L. Gastroenterology. 2004; 126: 1090-1103Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 9Kimmelman A.C. Qiao R.F. Narla G. Banno A. Lau N. Bos P.D. Nuñez Rodriguez N. Liang B.C. Guha A. Martignetti J.A. Friedman S.L. Chan A.M. Oncogene. 2004; 23: 5077-5083Crossref PubMed Scopus (67) Google Scholar, 10Ito G. Uchiyama M. Kondo M. Mori S. Usami N. Maeda O. Kawabe T. Hasegawa Y. Shimokata K. Sekido Y. Cancer Res. 2004; 64: 3838-3843Crossref PubMed Scopus (131) Google Scholar). Klf6 has been reported to be mutated in a large percentage of human prostate tumors (7Narla G. Heath K.E. Reeves H.L. Li D. Giono L.E. Kimmelman A.C. Glucksman M.J. Narla J. Eng F.J. Chan A.M. Ferrari A.C. Martignetti J.A. Friedman S.L. Science. 2001; 294: 2563-2566Crossref PubMed Scopus (377) Google Scholar), but its function during normal development has not been elucidated. Prostate development, specifically epithelial branching morphogenesis, is a well studied process. Outgrowth and branching of the prostate epithelial buds into the enveloping mesenchyme occur during the first 3 weeks postnatally (11Sugimura Y. Cunha G.R. Donjacour A.A. Biol. Reprod. 1986; 34: 961-971Crossref PubMed Scopus (311) Google Scholar). The fully developed prostate in an adult mouse is composed of three different paired lobes, referred as the anterior, ventral, and dorsal-lateral lobes. In addition, the number of main ducts and complexity of ductal branching vary among the three lobes. A number of growth factors/pathways have been implicated in regulating prostatic epithelial proliferation and differentiation, including hedgehog (Hh), 4The abbreviations used are: HhhedgehogBMPbone morphogenic proteinFGFfibroblastic growth factorShhsonic hedgehogMicro-CTmicro-computed tomographySMAsmooth muscle actinH&Ehematoxylin and eosinTCRDT-cell receptor delta chain. 4The abbreviations used are: HhhedgehogBMPbone morphogenic proteinFGFfibroblastic growth factorShhsonic hedgehogMicro-CTmicro-computed tomographySMAsmooth muscle actinH&Ehematoxylin and eosinTCRDT-cell receptor delta chain. bone morphogenic proteins (BMPs), fibroblastic growth factors (FGFs), and Notch and Wnt pathways (12Freestone S.H. Marker P. Grace O.C. Tomlinson D.C. Cunha G.R. Harnden P. Thomson A.A. Dev. Biol. 2003; 264: 352-362Crossref PubMed Scopus (128) Google Scholar, 13Wang B.E. Shou J. Ross S. Koeppen H. De Sauvage F.J. Gao W.Q. J. Biol. Chem. 2003; 278: 18506-18513Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 14Berman D.M. Desai N. Wang X. Karhadkar S.S. Reynon M. Abate-Shen C. Beachy P.A. Shen M.M. Dev. Biol. 2004; 267: 387-398Crossref PubMed Scopus (111) Google Scholar, 15Lamm M.L. Catbagan W.S. Laciak R.J. Barnett D.H. Hebner C.M. Gaffield W. Walterhouse D. Iannaccone P. Bushman W. Dev. Biol. 2002; 249: 349-366Crossref PubMed Scopus (137) Google Scholar, 16Thomson A.A. Cunha G.R. Development. 1999; 126: 3693-3701PubMed Google Scholar, 17Lamm M.L. Podlasek C.A. Barnett D.H. Lee J. Clemens J.Q. Hebner C.M. Bushman W. Dev. Biol. 2001; 232: 301-314Crossref PubMed Scopus (124) Google Scholar, 18Wang X.D. Leow C.C. Zha J. Tang Z. Modrusan Z. Radtke F. Aguet M. de Sauvage F.J. Gao W.Q. Dev. Biol. 2006; 290: 66-80Crossref PubMed Scopus (122) Google Scholar, 19Wang B.E. Wang X.D. Ernst J.A. Polakis P. Gao W.Q. PLoS ONE. 2008; 3: e2186Crossref PubMed Scopus (50) Google Scholar). For example, work done in our laboratory and others showed that sonic hedgehog (Shh) produced in the prostatic epithelium is a negative regulator of prostatic branching morphogenesis, mediated indirectly by periepithelial mesenchymal cells (12Freestone S.H. Marker P. Grace O.C. Tomlinson D.C. Cunha G.R. Harnden P. Thomson A.A. Dev. Biol. 2003; 264: 352-362Crossref PubMed Scopus (128) Google Scholar, 13Wang B.E. Shou J. Ross S. Koeppen H. De Sauvage F.J. Gao W.Q. J. Biol. Chem. 2003; 278: 18506-18513Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 14Berman D.M. Desai N. Wang X. Karhadkar S.S. Reynon M. Abate-Shen C. Beachy P.A. Shen M.M. Dev. Biol. 2004; 267: 387-398Crossref PubMed Scopus (111) Google Scholar, 15Lamm M.L. Catbagan W.S. Laciak R.J. Barnett D.H. Hebner C.M. Gaffield W. Walterhouse D. Iannaccone P. Bushman W. Dev. Biol. 2002; 249: 349-366Crossref PubMed Scopus (137) Google Scholar). In addition, while mesenchymal FGF10 stimulates prostatic epithelial growth (16Thomson A.A. Cunha G.R. Development. 1999; 126: 3693-3701PubMed Google Scholar), BMP4 is a mesenchymal factor that inhibits prostatic ductal budding and branching morphogenesis (17Lamm M.L. Podlasek C.A. Barnett D.H. Lee J. Clemens J.Q. Hebner C.M. Bushman W. Dev. Biol. 2001; 232: 301-314Crossref PubMed Scopus (124) Google Scholar). hedgehog bone morphogenic protein fibroblastic growth factor sonic hedgehog micro-computed tomography smooth muscle actin hematoxylin and eosin T-cell receptor delta chain. hedgehog bone morphogenic protein fibroblastic growth factor sonic hedgehog micro-computed tomography smooth muscle actin hematoxylin and eosin T-cell receptor delta chain. All Klf knockouts generated thus far are embryonic lethal (20Kuo C.T. Veselits M.L. Barton K.P. Lu M.M. Clendenin C. Leiden J.M. Genes Dev. 1997; 11: 2996-3006Crossref PubMed Scopus (304) Google Scholar, 21Nuez B. Michalovich D. Bygrave A. Ploemacher R. Grosveld F. Nature. 1995; 375: 316-318Crossref PubMed Scopus (476) Google Scholar, 22Segre J.A. Bauer C. Fuchs E. Nat. Genet. 1999; 22: 356-360Crossref PubMed Scopus (630) Google Scholar), including the Klf6 knock-out mice (23Matsumoto N. Kubo A. Liu H. Akita K. Laub F. Ramirez F. Keller G. Friedman S.L. Blood. 2006; 107: 1357-1365Crossref PubMed Scopus (101) Google Scholar). Because prostate development begins in late gestation and continues postnatally, we overcame embryonic lethality to study the role of Klf6 in prostate development and tumorigenesis by generating a prostate-specific deletion of the Klf6 gene using a Cre-lox recombination approach. We report here a novel role for Klf6 in the regulation of Shh-mediated epithelial branching morphogenesis in the anterior prostate. DISCUSSIONThe Klf gene family is known to be crucial for development in vertebrates (4Oates A.C. Pratt S.J. Vail B. Yan Y.I. Ho R.K. Johnson S.L. Postlethwait J.H. Zon L.I. Blood. 2001; 98: 1792-1801Crossref PubMed Scopus (92) Google Scholar). They are expressed in a large number of tissues (40Turner J. Crossley M. Trends Biochem. Sci. 1999; 24: 236-240Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar) and play an important role in the control of hematopoietic cell differentiation (4Oates A.C. Pratt S.J. Vail B. Yan Y.I. Ho R.K. Johnson S.L. Postlethwait J.H. Zon L.I. Blood. 2001; 98: 1792-1801Crossref PubMed Scopus (92) Google Scholar), erythroid cell maturation (21Nuez B. Michalovich D. Bygrave A. Ploemacher R. Grosveld F. Nature. 1995; 375: 316-318Crossref PubMed Scopus (476) Google Scholar, 41Perkins A.C. Gaensler K.M. Orkin S.H. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 12267-12271Crossref PubMed Scopus (127) Google Scholar), T-cell activation (42Kuo C.T. Veselits M.L. Leiden J.M. Science. 1997; 277: 1986-1990Crossref PubMed Scopus (342) Google Scholar), blood vessel stability (20Kuo C.T. Veselits M.L. Barton K.P. Lu M.M. Clendenin C. Leiden J.M. Genes Dev. 1997; 11: 2996-3006Crossref PubMed Scopus (304) Google Scholar), and skin permeability (22Segre J.A. Bauer C. Fuchs E. Nat. Genet. 1999; 22: 356-360Crossref PubMed Scopus (630) Google Scholar). Using the prostate as a model, we describe here for the first time a role for Klf6 in prostate branching morphogenesis. Consistent with this notion, Klf14, another Klf family member, has been shown to be critical for alveolarization in the developing lung. Deletion of Klf14 leads to thinner lung alveolar walls and poor outgrowth of secondary septa, which results in defective blood-air exchange and respiratory failures in newborn mice (43Hertveldt V. Louryan S. van Reeth T. Drèze P. van Vooren P. Szpirer J. Szpirer C. Dev. Dyn. 2008; 237: 883-892Crossref PubMed Scopus (26) Google Scholar). In the kidney, Klf6 is known to be expressed in the Wolffian duct, uteric bud, collecting ducts, and mesangium (44Fischer E.A. Verpont M.C. Garrett-Sinha L.A. Ronco P.M. Rossert J.A. J. Am Soc. Nephrol. 2001; 12: 726-735Crossref PubMed Google Scholar), an expression profile that would also be consistent with a role in branching morphogenesis.Because systemic loss of Klf6 is embryonic lethal (23Matsumoto N. Kubo A. Liu H. Akita K. Laub F. Ramirez F. Keller G. Friedman S.L. Blood. 2006; 107: 1357-1365Crossref PubMed Scopus (101) Google Scholar), we generated mice deficient for Klf6 specifically in the prostate. The resulting lateral branching defect in Klf6-deficient prostate was confirmed by light microscopy, H&E staining, and micro-CT imaging. Using a novel ex vivo ultrasound imaging modality, we were able to demonstrate that Klf6 plays a role in modulating the epithelial infolding and tufting, which results in decreased surface area of functional epithelium. The most prominent branching defects are seen in the anterior prostate, also known as a coagulating gland, which correlates with the highest level of Klf6 depletion (Fig. 1E). Despite the reduction in epithelial infolding and tufting whether or not prostatic function has been impaired in these mice remains to be determined.The augmented branching defect in the anterior prostate is noteworthy, because the branching patterns in the anterior prostate are distinct from the dorsolateral prostate and ventral prostate. In addition, the embryologic origin of the anterior prostate is also different from the other prostate lobes (45Cunha G.R. Lung B. Accesory Glands of Male Reproductive Tract. 6. Ann Arbor Science, Ann Arbor, MI1979: 1-28Google Scholar, 46Cunha G.R. Alarid E.T. Turner T. Donjacour A.A. Boutin E.L. Foster B.A. J. Androl. 1992; 13: 465-475PubMed Google Scholar). Although branching patterns of the ventral prostate and dorsolateral prostates are believed to arise from the urogenital sinus epithelium invaginating into urogenital sinus mesenchyme, the anterior prostate is thought to be derived from urogenital sinus epithelium at the most dorsal aspect of the urogenital sinus invaginating into the mesenchyme of the seminal vesicle, which itself is derived from the mesodermal Wolffian duct. Hence, the resulting branched structure in the anterior prostate has unique patterns distinct from the other prostate lobes and occurs as a result of lateral branch events and not as a result of bifurcations at the elongating tips.Consistent with previous reports from our group and others on the role of Shh signaling in prostate branching (12Freestone S.H. Marker P. Grace O.C. Tomlinson D.C. Cunha G.R. Harnden P. Thomson A.A. Dev. Biol. 2003; 264: 352-362Crossref PubMed Scopus (128) Google Scholar, 13Wang B.E. Shou J. Ross S. Koeppen H. De Sauvage F.J. Gao W.Q. J. Biol. Chem. 2003; 278: 18506-18513Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 14Berman D.M. Desai N. Wang X. Karhadkar S.S. Reynon M. Abate-Shen C. Beachy P.A. Shen M.M. Dev. Biol. 2004; 267: 387-398Crossref PubMed Scopus (111) Google Scholar, 15Lamm M.L. Catbagan W.S. Laciak R.J. Barnett D.H. Hebner C.M. Gaffield W. Walterhouse D. Iannaccone P. Bushman W. Dev. Biol. 2002; 249: 349-366Crossref PubMed Scopus (137) Google Scholar, 37Pu Y. Huang L. Prins G.S. Dev. Biol. 2004; 273: 257-275Crossref PubMed Scopus (89) Google Scholar), both Ptc-lacZ reporter mice crossed to Klf6f/fNkx3.1Cre/+ mice and in situ hybridization data in the present study indicate that impaired lateral branching in the Klf6-deficient prostate correlate with an up-regulation and loss of spatial localization of Shh, Ptc, and Gli1. Further support for the involvement of focal Hh signaling in prostate epithelial branching comes from a study by Bushman and co-workers (15Lamm M.L. Catbagan W.S. Laciak R.J. Barnett D.H. Hebner C.M. Gaffield W. Walterhouse D. Iannaccone P. Bushman W. Dev. Biol. 2002; 249: 349-366Crossref PubMed Scopus (137) Google Scholar), showing a close association between prostate ductal bud formation and localized expression of Shh at the growing tips of elongating prostate ducts. In addition, Shh regulates prostate branching morphogenesis in concert with FGF10 and BMP4 (37Pu Y. Huang L. Prins G.S. Dev. Biol. 2004; 273: 257-275Crossref PubMed Scopus (89) Google Scholar). Shh secreted from the epithelial ductal tips can down-regulate FGF10 that stimulates epithelial cell growth (16Thomson A.A. Cunha G.R. Development. 1999; 126: 3693-3701PubMed Google Scholar) and/or up-regulate BMP4 that inhibits epithelial cell growth (17Lamm M.L. Podlasek C.A. Barnett D.H. Lee J. Clemens J.Q. Hebner C.M. Bushman W. Dev. Biol. 2001; 232: 301-314Crossref PubMed Scopus (124) Google Scholar) in the mesenchyme, which results in epithelial ductal branching (37Pu Y. Huang L. Prins G.S. Dev. Biol. 2004; 273: 257-275Crossref PubMed Scopus (89) Google Scholar). In the present study, we show that loss of Klf6 leads to dispersed, rather than focal, Shh expression throughout the epithelial ducts and corresponding dispersed Ptc and Gli1 expression in the adjacent mesenchyme (Fig. 5). This broader domain of Hh pathway activity leads to an up-regulation of BMP4 expression, resulting in reduced ductal branching. As proposed by Pu and coworkers, the interplay and localized expression pattern of Shh and BMP4 are key to conveying critical branching information (37Pu Y. Huang L. Prins G.S. Dev. Biol. 2004; 273: 257-275Crossref PubMed Scopus (89) Google Scholar).Klf6 is reported to be a tumor suppressor that is inactivated in a large subset of prostate cancer (47Narla G. Friedman S.L. Martignetti J.A. Am. J. Pathol. 2003; 162: 1047-1052Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). However, our detailed histological characterization of Klf6f/fNkx3.1+/+ mice as old as 2 years failed to reveal any prostate tumors. 7C. C. Leow, B. Wang, J. Ross, S. M. Chan, J. Zha, R. A. D. Carano, G. Frantz, M. M. Shen, F. J. de Sauvage, and W.-Q. Gao, unpublished observations. The lack of tumor phenotype in the Klf6 mutant mice could be due to compensations/redundancy with other Klf family members. It is also possible that loss of Klf6 might not be sufficient to initiate prostate tumorigenesis in the mouse and that loss/mutation of additional tumor suppressors might be required. The Klf gene family is known to be crucial for development in vertebrates (4Oates A.C. Pratt S.J. Vail B. Yan Y.I. Ho R.K. Johnson S.L. Postlethwait J.H. Zon L.I. Blood. 2001; 98: 1792-1801Crossref PubMed Scopus (92) Google Scholar). They are expressed in a large number of tissues (40Turner J. Crossley M. Trends Biochem. Sci. 1999; 24: 236-240Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar) and play an important role in the control of hematopoietic cell differentiation (4Oates A.C. Pratt S.J. Vail B. Yan Y.I. Ho R.K. Johnson S.L. Postlethwait J.H. Zon L.I. Blood. 2001; 98: 1792-1801Crossref PubMed Scopus (92) Google Scholar), erythroid cell maturation (21Nuez B. Michalovich D. Bygrave A. Ploemacher R. Grosveld F. Nature. 1995; 375: 316-318Crossref PubMed Scopus (476) Google Scholar, 41Perkins A.C. Gaensler K.M. Orkin S.H. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 12267-12271Crossref PubMed Scopus (127) Google Scholar), T-cell activation (42Kuo C.T. Veselits M.L. Leiden J.M. Science. 1997; 277: 1986-1990Crossref PubMed Scopus (342) Google Scholar), blood vessel stability (20Kuo C.T. Veselits M.L. Barton K.P. Lu M.M. Clendenin C. Leiden J.M. Genes Dev. 1997; 11: 2996-3006Crossref PubMed Scopus (304) Google Scholar), and skin permeability (22Segre J.A. Bauer C. Fuchs E. Nat. Genet. 1999; 22: 356-360Crossref PubMed Scopus (630) Google Scholar). Using the prostate as a model, we describe here for the first time a role for Klf6 in prostate branching morphogenesis. Consistent with this notion, Klf14, another Klf family member, has been shown to be critical for alveolarization in the developing lung. Deletion of Klf14 leads to thinner lung alveolar walls and poor outgrowth of secondary septa, which results in defective blood-air exchange and respiratory failures in newborn mice (43Hertveldt V. Louryan S. van Reeth T. Drèze P. van Vooren P. Szpirer J. Szpirer C. Dev. Dyn. 2008; 237: 883-892Crossref PubMed Scopus (26) Google Scholar). In the kidney, Klf6 is known to be expressed in the Wolffian duct, uteric bud, collecting ducts, and mesangium (44Fischer E.A. Verpont M.C. Garrett-Sinha L.A. Ronco P.M. Rossert J.A. J. Am Soc. Nephrol. 2001; 12: 726-735Crossref PubMed Google Scholar), an expression profile that would also be consistent with a role in branching morphogenesis. Because systemic loss of Klf6 is embryonic lethal (23Matsumoto N. Kubo A. Liu H. Akita K. Laub F. Ramirez F. Keller G. Friedman S.L. Blood. 2006; 107: 1357-1365Crossref PubMed Scopus (101) Google Scholar), we generated mice deficient for Klf6 specifically in the prostate. The resulting lateral branching defect in Klf6-deficient prostate was confirmed by light microscopy, H&E staining, and micro-CT imaging. Using a novel ex vivo ultrasound imaging modality, we were able to demonstrate that Klf6 plays a role in modulating the epithelial infolding and tufting, which results in decreased surface area of functional epithelium. The most prominent branching defects are seen in the anterior prostate, also known as a coagulating gland, which correlates with the highest level of Klf6 depletion (Fig. 1E). Despite the reduction in epithelial infolding and tufting whether or not prostatic function has been impaired in these mice remains to be determined. The augmented branching defect in the anterior prostate is noteworthy, because the branching patterns in the anterior prostate are distinct from the dorsolateral prostate and ventral prostate. In addition, the embryologic origin of the anterior prostate is also different from the other prostate lobes (45Cunha G.R. Lung B. Accesory Glands of Male Reproductive Tract. 6. Ann Arbor Science, Ann Arbor, MI1979: 1-28Google Scholar, 46Cunha G.R. Alarid E.T. Turner T. Donjacour A.A. Boutin E.L. Foster B.A. J. Androl. 1992; 13: 465-475PubMed Google Scholar). Although branching patterns of the ventral prostate and dorsolateral prostates are believed to arise from the urogenital sinus epithelium invaginating into urogenital sinus mesenchyme, the anterior prostate is thought to be derived from urogenital sinus epithelium at the most dorsal aspect of the urogenital sinus invaginating into the mesenchyme of the seminal vesicle, which itself is derived from the mesodermal Wolffian duct. Hence, the resulting branched structure in the anterior prostate has unique patterns distinct from the other prostate lobes and occurs as a result of lateral branch events and not as a result of bifurcations at the elongating tips. Consistent with previous reports from our group and others on the role of Shh signaling in prostate branching (12Freestone S.H. Marker P. Grace O.C. Tomlinson D.C. Cunha G.R. Harnden P. Thomson A.A. Dev. Biol. 2003; 264: 352-362Crossref PubMed Scopus (128) Google Scholar, 13Wang B.E. Shou J. Ross S. Koeppen H. De Sauvage F.J. Gao W.Q. J. Biol. Chem. 2003; 278: 18506-18513Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 14Berman D.M. Desai N. Wang X. Karhadkar S.S. Reynon M. Abate-Shen C. Beachy P.A. Shen M.M. Dev. Biol. 2004; 267: 387-398Crossref PubMed Scopus (111) Google Scholar, 15Lamm M.L. Catbagan W.S. Laciak R.J. Barnett D.H. Hebner C.M. Gaffield W. Walterhouse D. Iannaccone P. Bushman W. Dev. Biol. 2002; 249: 349-366Crossref PubMed Scopus (137) Google Scholar, 37Pu Y. Huang L. Prins G.S. Dev. Biol. 2004; 273: 257-275Crossref PubMed Scopus (89) Google Scholar), both Ptc-lacZ reporter mice crossed to Klf6f/fNkx3.1Cre/+ mice and in situ hybridization data in the present study indicate that impaired lateral branching in the Klf6-deficient prostate correlate with an up-regulation and loss of spatial localization of Shh, Ptc, and Gli1. Further support for the involvement of focal Hh signaling in prostate epithelial branching comes from a study by Bushman and co-workers (15Lamm M.L. Catbagan W.S. Laciak R.J. Barnett D.H. Hebner C.M. Gaffield W. Walterhouse D. Iannaccone P. Bushman W. Dev. Biol. 2002; 249: 349-366Crossref PubMed Scopus (137) Google Scholar), showing a close association between prostate ductal bud formation and localized expression of Shh at the growing tips of elongating prostate ducts. In addition, Shh regulates prostate branching morphogenesis in concert with FGF10 and BMP4 (37Pu Y. Huang L. Prins G.S. Dev. Biol. 2004; 273: 257-275Crossref PubMed Scopus (89) Google Scholar). Shh secreted from the epithelial ductal tips can down-regulate FGF10 that stimulates epithelial cell growth (16Thomson A.A. Cunha G.R. Development. 1999; 126: 3693-3701PubMed Google Scholar) and/or up-regulate BMP4 that inhibits epithelial cell growth (17Lamm M.L. Podlasek C.A. Barnett D.H. Lee J. Clemens J.Q. Hebner C.M. Bushman W. Dev. Biol. 2001; 232: 301-314Crossref PubMed Scopus (124) Google Scholar) in the mesenchyme, which results in epithelial ductal branching (37Pu Y. Huang L. Prins G.S. Dev. Biol. 2004; 273: 257-275Crossref PubMed Scopus (89) Google Scholar). In the present study, we show that loss of Klf6 leads to dispersed, rather than focal, Shh expression throughout the epithelial ducts and corresponding dispersed Ptc and Gli1 expression in the adjacent mesenchyme (Fig. 5). This broader domain of Hh pathway activity leads to an up-regulation of BMP4 expression, resulting in reduced ductal branching. As proposed by Pu and coworkers, the interplay and localized expression pattern of Shh and BMP4 are key to conveying critical branching information (37Pu Y. Huang L. Prins G.S. Dev. Biol. 2004; 273: 257-275Crossref PubMed Scopus (89) Google Scholar). Klf6 is reported to be a tumor suppressor that is inactivated in a large subset of prostate cancer (47Narla G. Friedman S.L. Martignetti J.A. Am. J. Pathol. 2003; 162: 1047-1052Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). However, our detailed histological characterization of Klf6f/fNkx3.1+/+ mice as old as 2 years failed to reveal any prostate tumors. 7C. C. Leow, B. Wang, J. Ross, S. M. Chan, J. Zha, R. A. D. Carano, G. Frantz, M. M. Shen, F. J. de Sauvage, and W.-Q. Gao, unpublished observations. The lack of tumor phenotype in the Klf6 mutant mice could be due to compensations/redundancy with other Klf family members. It is also possible that loss of Klf6 might not be sufficient to initiate prostate tumorigenesis in the mouse and that loss/mutation of additional tumor suppressors might be required. We thank Leisa Johnson, Mallika Singh, Hua Tian, and Zhenyu Gu for critical discussions. We also thank Margaret Fuentes, Christine Olsson, and Luz Orellana for assistance with the animal colony, Allison Bruce with Graphics, Jeffrey Eastham-Anderson for Metamorph analysis of BMP4 in situ hybridization signal, and Howard Stern for his assistance with some histopathological review. Supplementary Material Download .pdf (2.09 MB) Help with pdf files Download .pdf (2.09 MB) Help with pdf files Prostate-specific Klf6 inactivation impairs anterior prostate branching morphogenesis through increased activation of the Shh pathway.Journal of Biological ChemistryVol. 286Issue 50PreviewVOLUME 284 (2009) PAGES 21057–21065 Full-Text PDF Open Access
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