We examined the effect of the potent MXC metabolite HPTE on global gene expression in cultured immature rat granulosa cells. This study shows that HPTE exerts a stronger inhibition on FSH-induced gene expression when compared to that regulated by exogenous cAMP. In addition, these data reveal for the first time the effects of HPTE on the expression of a plethora of genes which are important to numerous vital pathways in granulosa cells, including signal transduction, transport, cell differentiation, growth, survival, and apoptosis. We also analyzed the effect of HPTE on the expression of genes associated with ovarian function, showing that HPTE affects multiple genes previously linked with the processes of folliculogenesis, steroidogenesis, and/or ovulation.
exposure to MXC reduces serum progesterone levels and disrupts female reproductive parameters and ovarian morphology (Chapin et al., 1997
; Gray et al., 1989
). Transient developmental exposure to MXC can affect the level of key ovarian regulators including certain steroidogenic regulatory proteins (e.g., LHR and CYP11A1) (Armenti et al., 2008
). Direct inhibition of CYP11A1 enzyme activity by HPTE, leading to reduced progesterone production in cultured granulosa cells, was previously observed, although HPTE did not alter protein or mRNA levels of this enzyme (Akgul et al., 2008
). We have previously shown that HPTE inhibits FSH- and cAMP-induced steroid hormone production. Furthermore, we demonstrated HPTE-directed changes in the mRNA levels of several steroidogenic pathway proteins/enzymes in granulosa cells (Zachow and Uzumcu, 2006
). While HPTE completely abrogated CYP11A11
, and CYP19A1
mRNA levels in FSH-treated granulosa cells, HPTE reduced CYP19A1
mRNA levels by only 50% in the presence of cAMP (Zachow and Uzumcu, 2006
), indicating that FSH-stimulated cells were more sensitive to the inhibitory effects of HPTE. The current study confirmed that FSH-induced steroidogenesis is more sensitive to downregulation by HPTE. This action was maintained on a global scale as shown by the hierarchical clustering analyses. Hierarchical clustering profiles suggest that in the presence of FSH, effects manifest above 1μM HPTE, while granulosa cells given exogenous cAMP are more resistant to HPTE. The expression profile of FSH groups that were treated with 5 and 10μM of HPTE resembled the gene expression profile of the unstimulated (basal) groups. However, the expression profile of the cAMP-stimulated cells remained distinct. These data suggest that HPTE targets the cAMP-dependent cascade at one or more loci preceding cAMP production (e.g., FSH receptor, G proteins, and/or adenylyl cyclase) in immature rat granulosa cells. This is in contrast to a study by Chedrese and Feyles, where MXC had no observable effect on the levels of FSH-induced cAMP but still inhibited FSH-dependent steroid accumulation in porcine granulosa cells (Chedrese and Feyles, 2001
). This suggests that MXC exerts its effects distal to cAMP production. These apparently disparate results may be due to differences in action of MXC and HPTE in vitro
, specific culture conditions, and/or species-specific effects.
In the present report, FSH-treated granulosa cells were more sensitive to the effects of HPTE, and FSH represents a more physiological stimulus. Therefore, the FSH group was further analyzed to determine the HPTE-dependent alterations in gene expression. We examined those genes that exhibited the largest fold changes in expression and detected that many of these genes are associated with ovarian function (). HPTE suppressed the expression of several genes encoding mediators of steroidogenesis and/or ovulation (LHR, CYP11A1, HSD17B7, CYP19A1, FABP6, ACSBG1, PGR, and EGFR), transcription (SF-1, NR5A2, and CEBPA), and other aspects of ovarian function (KITL, INHβA, INHα, and PRLR). In contrast, HPTE upregulated the expression of several genes encoding mediators of apoptosis (CASP11, CASP12, and PDCD4), cell differentiation and growth regulation (GREM1, TGFβ2, TGFβ3, IGF-1, IGFBP1, and IGFBP5), ovulation/tissue remodeling (ADAMTS5), and stress response (DDIT3 and CA3).
Some genes of interest from this list are GREM1
(α subunit of inhibin), INHβA
subunit of inhibin), TGFB2
, and CASP12
. Gremlin 1 (GREM1
) expression was upregulated by HPTE. GREM1 is an antagonist of bone morphogenetic protein (BMP) signaling (Merino et al., 1999
) and is spatiotemporally expressed in the ovary (Pangas et al., 2004
). It is primarily expressed in granulosa cells within preantral and antral follicles. In large antral follicles, GREM1
mRNA is detected in cumulus but not in mural granulosa cells (Pangas et al., 2004
). While its expression is stimulated by BMPs and growth and differentiation factor-9 (GDF-9), GREM1 inhibits BMP signaling with no effect on GDF-9 signaling (Pangas et al., 2004
). Thus, it has been speculated that this regulated expression of GREM1 may inhibit the actions of theca cell-derived BMP on granulosa cell luteinization, while allowing GDF-9 of oocytic origin to mediate cumulus expansion (Pangas et al., 2004
). The fact that the expression of some of the luteinization markers (LHR and PGR EGFR) was inhibited by HPTE in the present study supports the above notion. Therefore, it maybe worth exploring the effect of HPTE on theca cell-derived BMPs and BMP-coupled signaling molecules.
In addition, GREM1 has been shown to affect other signaling pathways that may interact with the FSH/cAMP-dependent protein kinase A (PKA) signaling pathway. Overexpression of GREM1 has been observed to inhibit the activity of Wnt signaling through its connection with β-catenin (Gazzerro et al., 2007
). Recently, β-catenin was proven to be critical for gonadotropin-directed signal transduction (specifically FSH) through the coordination of SF-1 and β-catenin (Parakh et al., 2006
). Parakh et al. (2006)
also showed that β-catenin selectively modified the FSH-driven production of CYP19A1
in granulosa cells. In addition, β-catenin, in conjunction with SF-1, regulated the activity of CYP19A1 and was necessary for the FSH- and cAMP-mediated regulation of CYP19A1. In the present study, HPTE showed an inhibitory effect on the mRNAs encoding CYP19A1 and CYP11A1. This may be a result of a downstream effect on Wnt signaling via GREM1-induced β-catenin inhibition or other possible upstream effectors of β-catenin (Hino et al., 2005
). The Wnt pathway, and other signaling pathways that may interact with FSH-PKA signaling, appears to be one of many novel interactions that may be affected by HPTE in granulosa cells ().
FIG. 5. Proposed model for the effect of HPTE in the granulosa cell. FSH binding to the FSH receptor initiates the PKA signaling pathway that promotes E2 production in granulosa cells. HPTE appears to inhibit this cascade through an unknown mechanism, but effects (more ...)
Downregulation of the expression of both INHα
by HPTE may have significant consequences for the production of inhibins and activins in granulosa cells. Inhibins are dimers of an α subunit combined with either a βA
(inhibin A) or a βB
subunit (inhibin B) (Knight and Glister, 2006
). Thus, HPTE inhibition of INHα could affect the level of both inhibin isoforms. In addition, activins are homodimers (βA
, activin A, or βB
, activin B) or a heterodimer (activin AB) of β subunits (Knight and Glister, 2006
). Therefore, inhibition of INHβA
would also prevent formation of activins A and AB. Collectively, activins and inhibins have important roles in granulosa cell proliferation and differentiation. For example, activin stimulates basal and FSH-induced granulosa cell proliferation (Miro and Hillier, 1996
). Activin also regulates basal and gonadotropin-induced steroid production in rat granulosa cells (Miro et al., 1991
). A modulatory role of inhibin on follicular steroidogenesis has also been reported (Smyth et al., 1994
). As a result, inhibition of INHβA
may be causal to the reduced steroidogenesis in HPTE-treated granulosa cells (Zachow and Uzumcu, 2006
In addition to inhibin and activin, the expression of the mRNA encoding TGFβ2 and TGFβ3 was increased by HPTE. The TGFβs have a synergistic effect on FSH-stimulated proliferation in granulosa cells, and TGFβ-directed changes in granulosa cell steroidogenesis are well documented (Knight and Glister, 2006
). Since E2
has been shown to impair TGFβ expression (Kleuser et al., 2008
), a possible mechanism that explains the upregulation of TGFβs is the HPTE-dependent decrease in E2
and/or a direct effect of HPTE on TGFβ expression. However, further experiments are needed to determine this.
The long-chain fatty acid synthase known as Acyl-CoA synthase bubblegum 1 (ACSBG1
) was downregulated in granulosa cells challenged with HPTE. ACSBG1 is normally located in theca cells and testicular Leydig cells and has been linked to spermatogenesis (Pei et al., 2003
). The role of ACSBG1 in folliculogenesis has not been defined, but it may function as a survival factor, which mediates the synthesis of steroid precursors (Pei et al., 2003
). By inhibiting ACSBG1
, HPTE may reduce steroidogenesis by directly reducing steroid precursors and/or energy availability.
Numerous reports have shown the importance of the insulin-like growth factor (IGF) system within granulosa cells. In conjunction with FSH, IGF-I stimulates cell proliferation and steroidogenesis in granulosa cells of various species (deMoura et al., 1997
; Mazerbourg et al., 2003
). In contrast, insulin-like growth factor–binding proteins (IGFBPs) can suppress FSH-induced follicular growth and differentiation, leading to atresia by possibly sequestering IGF-I protein and inhibiting its activity (Cataldo et al., 1993
; Ui et al., 1989
). Interestingly, FSH reduces IGFBP activity by stimulating proteolytic mechanisms that degrade IGFBPs (Fielder et al., 1993
). In the present study, 10μM HPTE stimulated the genes encoding IGF-I and IGFBPs (i.e., IGFBP1 and IGFBP5) in FSH-stimulated granulosa cells. This appears to be controversial; however, the increase in IGF-I may counteract the HPTE-directed increase in the level of IGFBPs. In addition, E2
has been shown to inhibit IGFBP5 in granulosa cells from large follicles (Voge et al., 2004
); thus, the apparent upregulation of IGFBP5
by HPTE could be a consequence of the HPTE-dependent reduction in E2
production. In general, some of the effects on gene expression that were observed in HPTE-treated cells may be an indirect effect due to reduced E2
secretion as well as due to the direct effects of HPTE.
Others have noted that HPTE inhibits growth and induces atresia in antral follicles (Gupta et al., 2006
). HPTE upregulated caspases 11 and 12 mRNAs in granulosa cells, which may provide a mechanism that explains HPTE-induced atresia. Caspase 11 is classified as caspase 4 in humans and appears to be essential for activation of interleukin 1β–converting enzyme (caspase 1) (Wang et al., 1998
). Caspase 12 mediates apoptosis driven by the endoplasmic reticulum (Liu and Baliga, 2005
). Both caspases are upstream initiators of apoptosis through caspase 3 (Kang et al., 2000
; Liu and Baliga, 2005
). Whether HPTE affects caspases that regulate survival in granulosa cells is currently unknown but should be investigated.
Combining these observations, we have composed a working model that attempts to connect the observed gene expression changes in granulosa cells to some promising signaling mechanisms (). Evidence suggests that HPTE acts on the FSH-PKA signaling pathway (Zachow and Uzumcu, 2006
). Whether this interaction is direct or through still vaguely defined cross talk of parallel signaling cascades (Hunzicker-Dunn and Maizels, 2006
) is unknown. Multiple signaling complexes exist within granulosa cells, and each has some role in directing normal cellular function (Knight and Glister, 2006
). Our model attempts to link these pathways with inhibition of E2
production and steroidogenic pathway proteins/enzymes. Induction of GREM1
by HPTE is an example of multiple pathways that can be perturbed by HPTE. GREM1 is known to inhibit β-catenin; β-catenin is a proven stimulator of SF-1 leading to CYP19A1 activity (Gazzerro et al., 2005
; Michos et al., 2007
). Also, GREM1 binds to BMP and therefore would block BMP activity. This provides another potential mechanism for the inhibitory effects of HPTE in granulosa cells. Finally, Wnt functions through a pathway leading to β-catenin; so it is plausible that the effect of HPTE is also manifested through that cascade (Michos et al., 2007
). Since HPTE induced changes in the level of expression of several genes linked to granulosa cell signaling pathways, further efforts should be placed on determining the functional effects of the HPTE-stimulated alterations in gene expression.
In summary, the current results show that HPTE differentially affects FSH- and cAMP-stimulated gene expression in granulosa cells. In addition, these results indicate that parallel pathways, besides FSH-cAMP-PKA, may be involved in the effects of HPTE on FSH-mediated steroidogenesis in granulosa cells. The discovery of the HPTE-directed involvement of these and other pathways, and perhaps cross talk among numerous cascades, is compelling and will no doubt provide a better understanding of the effect of HPTE and MXC in the ovary.