We found consistent, characteristic differences in baseline gene expression patterns between genital skin fibroblasts from normal males and 46,XY female patients with AIS. Many of these differences between normal and AIS-derived fibroblasts were also observed in gonadal fibroblasts, suggesting that these differences are not purely due to differences in the anatomical site of origin of the fibroblasts. Interestingly, fibroblasts derived from abdominal and forearm skin, regions with relatively little sexual dimorphism, showed gene-expression patterns similar to the labial skin fibroblasts of AIS patients. Together, these data suggest that the AR is involved in determining a stable and stereotypical program of gene expression in genital fibroblasts that does not require the continuing presence of androgen for its maintenance.
A critical question raised by these results is whether the observed differences between genital fibroblasts from males and AIS females reflect cell-autonomous effects of androgen exposure during development, or indirect effects of the AR-dependent genital morphogenetic program. One possible interpretation of these data is that the distinct patterns of expression could have been due to differences in the origin or the developmental milieu of foreskin fibroblasts, derived from the genital tubercle, as opposed to the labial fibroblasts, derived from the genital swellings [10
]. The differences in gene expression we observed in AIS fibroblasts of gonadal origin compared to those of labial origin support this view (Figure ). We have observed consistent and characteristic differences in the gene-expression patterns of skin fibroblasts derived from different locations on the body [11
]. However, the current set of experiments suggests that the androgen receptor has a cell-autonomous role which contributes to a stable androgen-independent gene-expression pattern in genital fibroblasts. Expression patterns in cultured labial skin fibroblasts derived from two different individuals with AR mosaicism suggested that the cell-autonomous AR status was a relevant determinant of baseline gene expression in genital skin fibroblasts. Both these fibroblast lines, although derived from morphologically female genitalia in phenotypically female 46,XY individuals mosaic for AR-inactivating mutations, expressed wild-type AR in the cultured cells. These female AIS-affected individuals are thought to have acquired their AR
gene mutations post-zygotically [12
]. ARD364, which showed AR protein expression and binding in the range of normal male foreskin fibroblasts ([7
] and see Table ), despite its origin from anatomically female genitalia, had a gene-expression pattern indistinguishable from foreskin fibroblasts of normal males (Figure ). The second fibroblast line from an AR mosaic patient, ARD465 (Table ), had very low wild-type AR expression and showed baseline gene-expression patterns that were nevertheless more similar to normal male foreskin and prostate fibroblasts than to any of the AIS-derived cell lines (Figure ).
The discrepancy between the female phenotype of these mosaic individuals despite expression of the wild-type AR in cultured genital skin fibroblasts is not resolved to date [7
]. It may be explained by a time-dependent rise of an originally small fraction of cells containing the wild-type AR
allele in the mosaic genital mesenchyme during prenatal and postnatal development, or by differences between in vivo
and in vitro
conditions. Yet, the documented expression of the wild-type AR in cultures of these labial cells supports the idea that the AR status of the fibroblast was an important intrinsic determinant of the basal transcription patterns we identified. Therefore, the AR appears to be involved in setting long-lasting gene-expression patterns in genital skin fibroblasts.
Comparison of gene-expression patterns in genital fibroblasts from normal and AIS-affected individuals, and in fibroblasts from extragenital sites, may offer clues to the programs that underlie external genital development. Both cell adhesion and connective tissue remodeling are indispensable for normal development and maintenance of tissue integrity [13
]. The differential expression of proteoglycans, collagens and cell adhesion molecules (for example cadherin 13) might be involved in genital morphogenesis and later stability of sexually dimorphic traits of the external genitalia. Some genes expressed in wild-type AR cells could influence androgen signaling. For instance, aldo-keto reductase 1C1 is specifically involved in cellular androgen metabolism [16
] and thus may modulate the spectrum of cellular androgenic steroids available for activation of the AR. Structurally different androgens elicit different patterns of response from several androgen-responsive promoters, suggesting that the type of ligand present could affect cellular response [17
]. Mitogen-activated protein kinase 14, and STAT-induced STAT inhibitors 2 and 3 were expressed at significantly higher levels in cells with wild-type AR. Both MAP kinase and STAT pathways are involved in AR-dependent regulation and in ligand-independent activation of the AR [18
]. Differential expression of aldehyde dehydrogenase 1A1 and alcohol dehydrogenase 1B, enzymes that affect retinoic acid biosynthesis, suggest that other signaling pathways may participate in the AR-initiated programs of external genital differentiation [19
Several genes expressed specifically in the normal male foreskin fibroblasts have been previously implicated in male genital development, including HOXA13
, the T-box genes, BMP4
. Mutations in HOXA13
can cause distal limb and urogenital-tract malformations such as male hypospadias in hand-foot-genital syndrome [21
]. T-box genes (TBX
) are essential early regulators of limb development and also appear to be involved in male genital development [22
]. Mutations in TBX3
cause the ulnar-mammary syndrome characterized by limb, apocrine, and genital developmental abnormalities [23
]. Expression of T-box genes 2, 3, and 5 was significantly higher in normal male foreskin fibroblasts than in AIS genital fibroblasts. BMP4
has been implicated in ductal budding and branching during prostate development [24
] and a potential role of BMP4 in external genital development has also been postulated [25
has been found to have roles in sex-specific cell determination in the gonads and genital disc of Drosophila
]. Thus, mutations in genes characteristically expressed in normal male foreskin fibroblasts can, in some cases, lead to defective genital development. The data from these experiments therefore provide candidate genes for further investigation in patients with genital malformations.
As normal genital skin fibroblasts of 46,XY male individuals express the AR in vitro
(see Table and [4
]), we had anticipated that androgen treatment would elicit a transcriptional response program that could provide additional insights into the role of androgen in genital development. We have previously used a similar approach to delineate the transcriptional programs activated in prostate cancer cells in response to androgen [9
]. We had hoped that comparison of transcriptional responses of normal fibroblasts to those from AIS-affected individuals with varying degrees of genital ambiguity would provide still further insights into androgen's role in genital morphogenesis. However, we were unable to detect any significant changes in gene-expression patterns in cultured, AR-expressing genital fibroblasts or in AIS-derived fibroblasts in response to androgens. Although two previous reports have shown increases in aromatase enzymatic activity in genital skin fibroblasts treated with dihydrotestosterone (DHT) [8
], others have failed to observe changes in aromatase activity in response to androgen [28
]. In agreement with our findings, Elmlinger et al
. found significantly different baseline expression levels of insulin-like growth factor (IGF) and insulin-like growth factor binding protein (IGFBP) between normal and AIS-derived genital skin fibroblasts, and could not detect changes in transcript levels in response to androgen treatment [29
]. In normal genital fibroblasts, androgen-responsive reporter genes can only be activated by expression of co-transfected AR in the presence of ligand [30
]. Therefore, endogenous AR expression itself may be insufficient in genital skin fibroblasts to elicit a transcriptional response. Moreover, the lack of detectable changes in transcript levels for any of the 30,000 genes in the AIS-fibroblasts virtually excludes the possibility that DHT or R1881 could be acting through other steroid receptors or other signaling pathways.
The differences in androgen responsiveness we have observed between normal genital fibroblasts and prostate cancer epithelial cells in vitro
might reflect the responses seen in vivo
. Prostate epithelial cells retain exquisite sensitivity to androgen throughout life. Androgen deprivation produces profound involution of the prostate, particularly of the epithelial component, but little or no change in the external genitalia. It is possible that genital mesenchymal cells are only capable of responding to androgen at discrete stages in development in their specific in vivo
environment. In mice, stromal androgen responsiveness is restricted to the earliest stages in prostate development, and later the epithelial compartment becomes responsive and remains so [1
]. This responsiveness may be mediated through the expression of specific AR co-regulators. Compared to LNCaP cells, normal male genital fibroblasts show distinctly lower baseline expression of several AR co-regulators (such as NCOA2 (GRIP-1), NCOA3 (TRAM-1), ARA54 (RNF14), data not shown). Thus, genital fibroblasts may express critical AR co-regulators at discrete times during development that allow them to respond by setting up long-lasting transcriptional programs that underlie the genesis and maintenance of genital morphology.