Progenitor cells for the prostate epithelium: roles in development, regeneration, and cancer.

The identification of stem cell/progenitor populations represents a critical step for deducing the putative cell type(s) of origin for epithelial cancers and may provide important therapeutic insights. In the case of the prostate gland, recent studies have made significant progress in the identification of candidate stem cell populations, but they have left unresolved key questions about their tissue localization and functional properties. In our work, we have used genetic lineage marking in vivo to demonstrate that a rare epithelial cell population marked by expression of the Nkx3.1 homeobox gene in the androgen-deprived prostate contains bipotential progenitor cells that are capable of self-renewal. Inducible targeting of the Pten tumor suppressor in these castrate-resistant Nkx3.1-expressing cells demonstrates that this stem/progenitor population is also a potent cell of origin for prostate cancer in mouse models. These findings may help to explain several intriguing features of prostate cancer and its phenotypic progression.

BMP controls proximodistal outgrowth, via induction of the apical ectodermal ridge, and dorsoventral patterning in the vertebrate limb.

Dorsoventral (DV) patterning of the vertebrate limb requires the function of the transcription factor Engrailed 1 (EN1) in the ventral ectoderm. EN1 restricts, to the dorsal half of the limb, the expression of the two genes known to specify dorsal pattern. Limb growth along the proximodistal (PD) axis is controlled by the apical ectodermal ridge (AER), a specialized epithelium that forms at the distal junction between dorsal and ventral ectoderm. Using retroviral-mediated misexpression of the bone morphogenetic protein (BMP) antagonist Noggin or an activated form of the BMP receptor in the chick limb, we demonstrate that BMP plays a key role in both DV patterning and AER induction. Thus, the DV and PD axes are linked by a common signal. Loss and gain of BMP function experiments show that BMP signaling is both necessary and sufficient to regulate EN1 expression, and consequently DV patterning. Our results also indicate that BMPs are required during induction of the AER. Manipulation of BMP signaling results in either disruptions in the endogenous AER, leading to absent or severely truncated limbs or the formation of ectopic AERs that can direct outgrowth. Moreover, BMP controls the expression of the MSX transcription factors, and our results suggest that MSX acts downstream of BMP in AER induction. We propose that the BMP signal bifurcates at the level of EN1 and MSX to mediate differentially DV patterning and AER induction, respectively.

Crystal structure of the Msx-1 homeodomain/DNA complex.

The Msx-1 homeodomain protein plays a crucial role in craniofacial, limb, and nervous system development. Homeodomain DNA-binding domains are comprised of 60 amino acids that show a high degree of evolutionary conservation. We have determined the structure of the Msx-1 homeodomain complexed to DNA at 2.2 A resolution. The structure has an unusually well-ordered N-terminal arm with a unique trajectory across the minor groove of the DNA. DNA specificity conferred by bases flanking the core TAAT sequence is explained by well ordered water-mediated interactions at Q50. Most interactions seen at the TAAT sequence are typical of the interactions seen in other homeodomain structures. Comparison of the Msx-1-HD structure to all other high resolution HD-DNA complex structures indicate a remarkably well-conserved sphere of hydration between the DNA and protein in these complexes.

Msx homeobox genes inhibit differentiation through upregulation of cyclin D1.

During development, patterning and morphogenesis of tissues are intimately coordinated through control of cellular proliferation and differentiation. We describe a mechanism by which vertebrate Msx homeobox genes inhibit cellular differentiation by regulation of the cell cycle. We show that misexpression of Msx1 via retroviral gene transfer inhibits differentiation of multiple mesenchymal and epithelial progenitor cell types in culture. This activity of Msx1 is associated with its ability to upregulate cyclin D1 expression and Cdk4 activity, while Msx1 has minimal effects on cellular proliferation. Transgenic mice that express Msx1 under the control of the mouse mammary tumor virus long terminal repeat (MMTV LTR) display impaired differentiation of the mammary epithelium during pregnancy, which is accompanied by elevated levels of cyclin D1 expression. We propose that Msx1 gene expression maintains cyclin D1 expression and prevents exit from the cell cycle, thereby inhibiting terminal differentiation of progenitor cells. Our model provides a framework for reconciling the mutant phenotypes of Msx and other homeobox genes with their functions as regulators of cellular proliferation and differentiation during embryogenesis.

Roles for Msx and Dlx homeoproteins in vertebrate development.

This review provides a comparative analysis of the expression patterns, functions, and biochemical properties of Msx and Dlx homeobox genes. These comprise multi-gene families that are closely related with respect to sequence features as well as expression patterns during vertebrate development. Thus, members of the Msx and Dlx families are expressed in overlapping, but distinct, patterns and display complementary or antagonistic functions, depending upon the context. A common theme shared among Msx and Dlx genes is that they are required during early, middle, and late phases of development where their differential expression mediates patterning, morphogenesis, and histogenesis of tissues in which they are expressed. With respect to their biochemical properties, Msx proteins function as transcriptional repressors, while Dlx proteins are transcriptional activators. Moreover, their ability to oppose each other's transcriptional actions implies a mechanism underlying their complementary or antagonistic functions during development.

A novel PF/PN motif inhibits nuclear localization and DNA binding activity of the ESX1 homeoprotein.

Despite their significance for mammalian embryogenesis, the molecular mechanisms that regulate placental growth and development have not been well defined. The Esx1 homeobox gene is of particular interest because it is among the few regulatory genes that have specific expression and function in the placenta during murine development. In addition, the ESX1 protein contains several notable features that are not often associated with homeoproteins, including an atypical homeodomain of the paired-like class, a proline-rich region that contains an SH3 binding motif, and a novel repeat region consisting of prolines alternating with phenylalanines or asparagines that we term the PF/PN motif. We have found that the ESX1 protein is expressed in the labyrinth layer of the placenta in vivo, where its subcellular localization is primarily cytoplasmic. Our results suggest that this unexpected subcellular localization is conferred by the PF/PN motif, which inhibits nuclear localization of ESX1 in cell culture, as well as its DNA binding activity in vitro. Finally, we show that the proline-rich region of ESX1 mediates interactions in vitro with the c-abl SH3 domain as well as with certain WW domains. We propose that the PF/PN motif provides a novel mechanism for regulating nuclear entry and that the essential function of ESX1 during placental development is mediated by its ability to couple cytoplasmic signal transduction events with transcriptional regulation in the nucleus.

Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors.

The migration of myogenic precursors to the vertebrate limb exemplifies a common problem in development - namely, how migratory cells that are committed to a specific lineage postpone terminal differentiation until they reach their destination. Here we show that in chicken embryos, expression of the Msx1 homeobox gene overlaps with Pax3 in migrating limb muscle precursors, which are committed myoblasts that do not express myogenic differentiation genes such as MyoD. We find that ectopic expression of Msx1 in the forelimb and somites of chicken embryos inhibits MyoD expression as well as muscle differentiation. Conversely, ectopic expression of Pax3 activates MyoD expression, while co-ectopic expression of Msx1 and Pax3 neutralizes their effects on MyoD. Moreover, we find that Msx1 represses and Pax3 activates MyoD regulatory elements in cell culture, while in combination, Msx1 and Pax3 oppose each other's trancriptional actions on MyoD. Finally, we show that the Msx1 protein interacts with Pax3 in vitro, thereby inhibiting DNA binding by Pax3. Thus, we propose that Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors via direct protein-protein interaction. Our results implicate functional antagonism through competitive protein-protein interactions as a mechanism for regulating the differentiation state of migrating cells.

Roles for Nkx3.1 in prostate development and cancer.

In aging men, the prostate gland becomes hyperproliferative and displays a propensity toward carcinoma. Although this hyperproliferative process has been proposed to represent an inappropriate reactivation of an embryonic differentiation program, the regulatory genes responsible for normal prostate development and function are largely undefined. Here we show that the murine Nkx3.1 homeobox gene is the earliest known marker of prostate epithelium during embryogenesis and is subsequently expressed at all stages of prostate differentiation in vivo as well as in tissue recombinants. A null mutation for Nkx3.1 obtained by targeted gene disruption results in defects in prostate ductal morphogenesis and secretory protein production. Notably, Nkx3.1 mutant mice display prostatic epithelial hyperplasia and dysplasia that increases in severity with age. This epithelial hyperplasia and dysplasia also occurs in heterozygous mice, indicating haploinsufficiency for this phenotype. Because human NKX3.1 is known to map to a prostate cancer hot spot, we propose that NKX3.1 is a prostate-specific tumor suppressor gene and that loss of a single allele may predispose to prostate carcinogenesis. The Nkx3.1 mutant mice provide a unique animal model for examining the relationship between normal prostate differentiation and early stages of prostate carcinogenesis.

Molecular biology of prostate development and prostate cancer.

The molecular mechanisms that control prostate development have been intensely studied in recent years due to the emergence of prostatic cancer as a major health concern. Several recent studies have led to the identification of numerous genes that are required for prostate organogenesis, many of which also contribute to prostate carcinogenesis. These genes fall into several categories, including proto-oncogenes, transcription factors, homeobox genes, growth factors and cell adhesion molecules. This review focuses on those genes which have been implicated in prostate growth and development, and which exhibit deregulated expression in prostate cancer.

An early phase of embryonic Dlx5 expression defines the rostral boundary of the neural plate.

Relatively little is known about the molecular events that specify the rostrocaudal axis of the neural plate. Here we show that a member of the Distal-less (Dlx) homeobox gene family, Dlx5, is one of the earliest known markers for the most rostral ectoderm, before the formation of an overt neural plate. During late gastrulation Dlx5 expression becomes localized to the anterior neural ridge, which defines the rostral boundary of the neural plate, and also extends caudolaterally, marking the region of the presumptive neural crest. Subsequently, Dlx5 is expressed in tissues (olfactory epithelium, ventral cephalic epithelium) that are believed to derive from the anterior neural ridge, based on the avian fate map. The early phase of Dlx5 expression in the anterior neural ridge and its derivatives is distinct from a later phase of expression in the ventral telencephalon and diencephalon and also appears to be unique for Dlx5 among members of the Dlx family. Another distinctive feature of Dlx5 expression is the occurrence of an alternative transcript (deltaDlx5), which encodes a truncated protein lacking the homeodomain, and represents a significant fraction of total Dlx5 transcripts at all embryonic stages that were examined. In contrast with full-length DLX5, the deltaDLX5 truncated protein is deficient in DNA-binding activity and does not interact with the homeoprotein partner MSX1. Taken together, our findings suggest that Dlx5 activity may be regulated via the expression of an alternative transcript and demonstrate that Dlx5 marks the anterior boundary of the neural plate.

Haploinsufficiency of MSX1: a mechanism for selective tooth agenesis.

Previously, we found that the cause of autosomal dominant selective tooth agenesis in one family is a missense mutation resulting in an arginine-to-proline substitution in the homeodomain of MSX1. To determine whether the tooth agenesis phenotype may result from haploinsufficiency or a dominant-negative mechanism, we have performed biochemical and functional analyses of the mutant protein Msx1(R31P). We show that Msx1(R31P) has perturbed structure and reduced thermostability compared with wild-type Msx1. As a consequence, the biochemical activities of Msx1(R31P) are severely impaired, since it exhibits little or no ability to interact with DNA or other protein factors or to function in transcriptional repression. We also show that Msx1(R31P) is inactive in vivo, since it does not display the activities of wild-type Msx1 in assays of ectopic expression in the limb. Furthermore, Msx1(R31P) does not antagonize the activity of wild-type Msx1 in any of these assays. Because Msx1(R31P) appears to be inactive and does not affect the action of wild-type Msx1, we propose that the phenotype of affected individuals with selective tooth agenesis is due to haploinsufficiency.

Protein complex formation between Msx1 and Lhx2 homeoproteins is incompatible with DNA binding activity.

Msx genes encode a family of homeoproteins that function as transcription repressors through protein-protein interactions. Here we show that Lhx2, a LIM-type homeoprotein, is a protein partner for Msx1 in vitro and in cellular extracts. The interaction between Msx1 and Lhx2 is mediated through the homeodomain-containing regions of both proteins. Interestingly, the LIM domains, which serve as protein interaction domains for other partners of Lhx2, are not required for the Msx1-Lhx2 association. We show that Msx1 and Lhx2 form a protein complex in the absence of DNA, and that DNA binding by either protein alone can occur at the expense of protein complex formation. The significance of this protein-protein interaction is underscored by the expression patterns of Msx1 and Lhx2, which are partially overlapping during murine embryogenesis. The description of Lhx2 as a protein partner for Msx1 suggests that the functional specificity of homeoproteins in vivo is determined by a balance between their association with DNA and their protein partners.

A novel human prostate-specific, androgen-regulated homeobox gene (NKX3.1) that maps to 8p21, a region frequently deleted in prostate cancer.

We have isolated a prostate-specific gene (NKX3.1) in humans that is homologous to the Drosophila NK homeobox gene family. Northern blot analyses indicate that this gene is expressed at high levels in adult prostate and at a much lower level in testis, but is expressed little or not at all in several other tissues. In an androgen-dependent prostate carcinoma line, LNCaP, NKX3.1 mRNA is expressed at a basal level that was increased markedly upon androgen stimulation; the NKX3.1 mRNA was undetectable in several other human tumor cell lines including two androgen-independent prostate carcinoma lines. The NKX3.1 gene maps to chromosome band 8p21, a region frequently reported to undergo a loss of heterozygosity associated with tissue dedifferentiation and loss of androgen responsiveness during the progression of prostate cancer. Based on these data we propose that NKX3.1 is a candidate gene for playing a role in the opposing processes of androgen-driven differentiation of prostatic tissue and loss of that differentiation during the progression of prostate cancer.

Homology modeling using simulated annealing of restrained molecular dynamics and conformational search calculations with CONGEN: application in predicting the three-dimensional structure of murine homeodomain Msx-1.

We have developed an automatic approach for homology modeling using restrained molecular dynamics and simulated annealing procedures, together with conformational search algorithms available in the molecular mechanics program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26:137-168). The accuracy of the method is validated by "predicting" structures of two homeodomain proteins with known three-dimensional structures, and then applied to predict the three-dimensional structure of the homeodomain of the murine Msx-1 transcription factor. Regions of the unknown protein structure that are highly homologous to the known template structure are constrained by "homology distance constraints," whereas the conformations of nonhomologous regions of the unknown protein are defined only by the potential energy function. A full energy function (excluding explicit solvent) is employed to ensure that the calculated structures have good conformational energies and are physically reasonable. As in NMR structure determinations, information on the consistency of the structure prediction is obtained by superposition of the resulting family of protein structures. In this paper, our homology modeling algorithm is described and compared with related homology modeling methods using spatial constraints derived from the structures of homologous proteins. The software is then used to predict the DNA-bound structures of three homeodomain proteins from the X-ray crystal structure of the engrailed homeodomain protein (Kissinger CR et al., 1990, Cell 63:579-590). The resulting backbone and side-chain conformations of the modeled yeast Mat alpha 2 and D. melanogaster Antennapedia homeodomains are excellent matches to the corresponding published X-ray crystal (Wolberger C et al., 1991, Cell 67:517-528) and NMR (Billeter M et al., 1993, J Mol Biol 234:1084-1097) structures, respectively. Examination of these structures of Msx-1 reveals a network of highly conserved surface salt bridges that are proposed to play a role in regulating protein-protein interactions of homeodomains in transcription complexes.

Tissue-specific expression of murine Nkx3.1 in the male urogenital system.

The molecular mechanisms involved in growth and morphogenesis of the mammalian urogenital system are largely undefined. In this study, we describe the cloning and characterization of a novel murine homeobox gene, Nkx3.1, which is expressed in the male urogenital system during late embryogenesis and adulthood. We show that Nkx3.1 encodes a 38 kDa homeoprotein that has DNA binding properties similar to those of other Nkx family members. By RNAse protection analysis, we demonstrate that Nkx3.1 is expressed in late-gestation embryos and adults by tissues of the male urogenital system, including the testis, seminal vesicle, and the prostate. In adult males, expression of Nkx3.1 in the prostate increases during sexual maturation, and is significantly reduced following castration, suggesting that androgens are required for maintenance of Nkx3.1 expression. In situ hybridization analysis of mid- and late-gestation male embryos shows that Nkx3.1 is expressed in the developing urogenital sinus, testis, and prostatic buds. In addition to its expression in the urogenital system, we also find that Nkx3.1 is expressed in the dorsal aorta and kidney. These results implicate Nkx3.1 in the growth and development of the prostate and/or other tissues of the male urogenital system, and suggest that Nkx3.1 may play a role in sexually dimorphic as well as non-sexually dimorphic organogenesis.

Heterodimerization of Msx and Dlx homeoproteins results in functional antagonism.

Protein-protein interactions are known to be essential for specifying the transcriptional activities of homeoproteins. Here we show that representative members of the Msx and Dlx homeoprotein families form homo- and heterodimeric complexes. We demonstrate that dimerization by Msx and Dlx proteins is mediated through their homeodomains and that the residues required for this interaction correspond to those necessary for DNA binding. Unlike most other known examples of homeoprotein interactions, association of Msx and Dlx proteins does not promote cooperative DNA binding; instead, dimerization and DNA binding are mutually exclusive activities. In particular, we show that Msx and Dlx proteins interact independently and noncooperatively with homeodomain DNA binding sites and that dimerization is specifically blocked by the presence of such DNA sites. We further demonstrate that the transcriptional properties of Msx and Dlx proteins display reciprocal inhibition. Specifically, Msx proteins act as transcriptional repressors and Dlx proteins act as activators, while in combination, Msx and Dlx proteins counteract each other's transcriptional activities. Finally, we show that the expression patterns of representative Msx and Dlx genes (Msx1, Msx2, Dlx2, and Dlx5) overlap in mouse embryogenesis during limb bud and craniofacial development, consistent with the potential for their protein products to interact in vivo. Based on these observations, we propose that functional antagonism through heterodimer formation provides a mechanism for regulating the transcriptional actions of Msx and Dlx homeoproteins in vivo.

Rapid identification of homeodomain binding sites in the Wnt-5a gene using an immunoprecipitation strategy.

Here we describe an immunoprecipitation approach for identifying homeodomain binding sites within uncharacterized genomic sequences of a putative downstream target gene, Wnt-5a. Immunoprecipitation of Wnt-5a genomic fragments was performed using a purified Msx1 homeodomain polypeptide (Msx1) and its corresponding antisera (alpha-Msx1). This resulted in isolation of three fragments containing multiple DNA binding sites for Msx1, as confirmed by DNA binding studies. The three fragments were contiguous within a 3.4 kb intronic sequence of Wnt-5a. Moreover, at least one of the Msx1 sites has been conserved throughout evolution, suggesting that these sites may comprise or contribute to a regulatory element for Wnt-5a. We propose that the immunoprecipitation strategy permits a rapid, initial approach for identifying functionally-relevant homeodomain binding sites within target genes whose regulatory sequences have not yet been previously elucidated.

Repression by HoxA7 is mediated by the homeodomain and the modulatory action of its N-terminal-arm residues.

Hox genes encode homeodomain-containing proteins that are presumed to control spatial patterning during murine embryogenesis through their actions as transcriptional regulatory proteins. In this study, we have investigated the transcriptional function of a prototypic member of this family, HoxA7. We demonstrate that HoxA7 function as a potent transcriptional repressor and that its action as such requires several domains, including both activator and repressor regions. The repressor regions are contained within the homeodomain and a C-terminal acidic region, both of which are well conserved among members of the Hox family. Accordingly, we show that two other members of this family also function as repressors, although they vary in their relative repressor potency. Finally, we explore the novel observation that the homeodomain of HoxA7 functions as a transcriptional repressor domain. We show that the homeodomain compared with two other DNA-binding domains, is unique in its ability to function as a repressor domain and that repression requires conserved residues, in helix III. We further show that residues in the N-terminal arm of the homeodomain contribute to the differential repressor actions of various Hox proteins. These findings demonstrate that the transcriptional function of HoxA7 and possibility of Hox proteins in general is determined by their unique combination of conserved and nonconserved regions as well as through the complex actions of their homeodomains.