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Biomarkers; Colorectal cancer; Maintenance therapy Biomarcadors; Càncer colorectal; Teràpia de manteniment Biomarcadores; Cáncer colorrectal; Terapia de mantenimiento Purpose MODUL is an adaptable, signal-seeking trial of biomarker-driven maintenance therapy following first-line induction treatment in patients with metastatic colorectal cancer (mCRC). We report findings from Cohorts 1 (BRAFmut), 3 (human epidermal growth factor 2 [HER2]+) and 4 (HER2‒/high microsatellite instability, HER2‒/microsatellite stable [MSS]/BRAFwt or HER2‒/MSS/BRAFmut/RASmut). Methods Patients with unresectable, previously untreated mCRC without disease progression following standard induction treatment (5-fluorouracil/leucovorin [5-FU/LV] plus oxaliplatin plus bevacizumab) were randomly assigned to control (fluoropyrimidine plus bevacizumab) or cohort-specific experimental maintenance therapy (Cohort 1: vemurafenib plus cetuximab plus 5-FU/LV; Cohort 3: capecitabine plus trastuzumab plus pertuzumab; Cohort 4: cobimetinib plus atezolizumab). The primary efficacy end-point was progression-free survival (PFS). Results Cohorts 1, 3 and 4 did not reach target sample size because of early study closure. In Cohort 1 (n = 60), PFS did not differ between treatment arms (hazard ratio, 0.95; 95% confidence intervals 0.50–1.82; P = 0.872). However, Cohort 1 exploratory biomarker data showed preferential selection for mitogen-activated protein kinase (MAPK) pathway mutations (mainly KRAS, NRAS, MAP2K1 or BRAF) in the experimental arm but not the control arm. In Cohort 3 (n = 5), PFS ranged from 3.6 to 14.7 months versus 4.0 to 5.4 months in the experimental and control arms, respectively. In Cohort 4 (n = 99), PFS was shorter in the experimental arm (hazard ratio, 1.44; 95% confidence intervals 0.90–2.29; P = 0.128). Conclusions Vemurafenib plus cetuximab plus 5-FU/LV warrants further investigation as first-line maintenance treatment for BRAFmut mCRC. MAPK-pathway emergent genomic alterations may offer novel therapeutic opportunities in BRAFmut mCRC. Cobimetinib plus atezolizumab had an unfavourable benefit:risk ratio in HER2‒/MSS/BRAFwt mCRC. New strategies are required to increase the susceptibility of MSS mCRC to immunotherapy. This work was supported by F. Hoffmann-La Roche Ltd.

Summary: Correct positioning of T cells within infected tissues is critical for T cell activation and pathogen control. Upon tissue entry, effector T cells must efficiently locate antigen-presenting cells (APC) for peripheral activation. We reveal that tissue entry and initial peripheral activation of Th1 effector T cells are tightly linked to perivascular positioning of chemokine-expressing APCs. Dermal inflammation induces tissue-wide de novo generation of discrete perivascular CXCL10+ cell clusters, enriched for CD11c+MHC-II+ monocyte-derived dendritic cells. These chemokine clusters are “hotspots” for both Th1 extravasation and activation in the inflamed skin. CXCR3-dependent Th1 localization to the cluster micro-environment prolongs T-APC interactions and boosts function. Both the frequency and range of these clusters are enhanced via a T helper 1 (Th1)-intrinsic, interferon-gamma (IFNγ)-dependent positive-feedback loop. Thus, the perivascular CXCL10+ clusters act as initial peripheral activation niches, optimizing controlled activation broadly throughout the tissue by coupling Th1 tissue entry with enhanced opportunities for Th1-APC encounter.

Pathogen control requires T-cells to locate and make direct contact with antigen-presenting cells (APCs) for peripheral reactivation and delivery of effector molecules. The signals that orchestrate T-cell-APC interactions are poorly understood in inflamed peripheral tissues. Using intravital imaging (IVI), we have found that Th1 cell entry into the inflamed dermis is nucleated by perivascular CXCL10+ cellular clusters enriched for APCs. Th1 cells form more stable interactions with APCs within these clusters compared to outer regions. CXCL10+ clusters are amplified in a IFNγ-dependent manner, where CD4+ T-cell IFNγ release increases APC recruitment and CXCL10 expression. We hypothesize that these chemokine clusters serve as CXCL10 Peripheral Activation (PAC-10) niches, where Th1 cells achieve early peripheral activation away from the pathogen modulated milieu. We seek to determine the relationship between these spatially restricted PAC-10 niches, infection foci, and pathogen clearance. Using a combination of IVI, flow cytometry, and transcriptomics we characterized niche specific Th1 cells and niche cellular composition to determine if PAC-10 niches modulate Th1 cells for pathogen control. Initial studies reveal that niche specific Th1 cells have increased CD69 and IFNγ expression compared to non-niche counterparts. PAC-10 niches appear to have a spatially distinct innate cell population, likely with enhanced ability to support Th1 activation. Future studies will characterize the functional status of niche specific Th1 cells and evaluate the necessity of PAC-10 niches for pathogen control. These studies will inform therapeutic strategies to boost T-cell activation in infection or hinder activation in autoimmunity. Supported by NIH NIAID P01 AI02851 NIH NIAID R01 AI070826

SUMMARYCorrect positioning of T cells within infected tissues is critical for T cell activation and pathogen control. Upon tissue entry, effector T cells must efficiently locate antigen presenting cells (APC) for peripheral activation. We reveal that tissue entry and initial peripheral activation of Th1 effector T cells are tightly linked to perivascular positioning of chemokine-expressing APCs. Dermal inflammation induced tissue-wide de novo generation of discrete perivascular CXCL10+ cell clusters, enriched for CD11c+MHC-II+ monocyte-derived dendritic cells. These chemokine clusters were ‘hot spots’ for both Th1 extravasation and activation in the inflamed skin. CXCR3-dependent Th1 localization to the cluster micro-environment prolonged T-APC interactions and boosted function. Both the frequency and range of these clusters were enhanced via a Th1-intrinsic, IFNγ-dependent positive feedback loop. Thus, the perivascular CXCL10+ clusters act as initial peripheral activation niches, optimizing controlled activation broadly throughout the tissue by coupling Th1 tissue entry with enhanced opportunities for Th1-APC encounter.

Correct positioning of T cells within infected tissues is critical for T cell activation and pathogen control. Upon tissue entry, effector T cells must efficiently locate antigen-presenting cells (APC) for peripheral activation. We reveal that tissue entry and initial peripheral activation of Th1 effector T cells are tightly linked to perivascular positioning of chemokine-expressing APCs. Dermal inflammation induces tissue-wide de novo generation of discrete perivascular CXCL10

A substantial amount of evidence suggests that androgen signaling through classical androgen receptors is critical for both normal and pathologic ovarian physiology. Specifically, we and others have shown that, in mouse granulosa cells, androgen actions through both extranuclear and nuclear androgen receptor signaling are critical for normal follicle development and ovulation. Here, we show that androgens through the PI3K/Akt pathway rapidly (within minutes) phosphorylate and inhibit activity of the Polycomb group protein enhancer of zeste homolog 2 (Ezh2). Over the course of 24 to 48 hours, androgens then induce expression of the microRNA miR-101, which targets Ezh2 messenger RNA (mRNA), leading to a nearly complete loss of Ezh2 protein expression. This long-term androgen-induced loss of Ezh2 actions ultimately results in sustained reduction of the H3K27me3-repressive mark in the promoter region of the Runt-related transcription factor-1 (Runx1) gene, a luteinizing hormone (LH)–induced transcription factor essential for ovulation, leading to increased Runx1 mRNA expression. Accordingly, blocking androgen-induced inhibition of Ezh2 in vivo adversely affects LH-induced Runx1 mRNA expression and subsequent ovulation. Importantly, although estrogen treatment of granulosa cells similarly causes rapid activation of the PI3K/Akt pathway and short-term phosphorylation of Ezh2, it does not induce miR-101 expression and thereby does not reduce overall Ezh2 expression, demonstrating the androgen specificity of long-term Ezh2 suppression. Thus, this study provides insight regarding how androgen-induced extranuclear kinase signaling and intranuclear transcription through Ezh2 modifications may influence the expression pattern of genes, ultimately affecting various downstream physiological processes.

Lymphangioleiomyomatosis (LAM) is a rare disease occurring almost exclusively in women whereby metastatic smooth-muscle cell-like adenomas grow within the lungs, resulting in loss of pulmonary function. LAM cells contain mutations in tuberous sclerosis 1 or 2 genes (TSC1 or TSC2), which leads to heightened mammalian target of rapamycin complex1 (mTORC1) activity and cell proliferation. The LAM cell origin remains unknown; however, we reported that inactivation of the Tsc2 gene in the mouse uterus resulted in myometrial tumors with nearly all known features of LAM, including over-expression of melanocytic markers that are pathognomonic of LAM tumor cells. Approximately 50% of animals developed metastatic myometrial tumors in the lung, suggesting that LAM cells might originate from the uterine myometrium, possibly explaining its overwhelming female prevalence. Estrogen ablation by oophorectomy or aromatase inhibition resulted in nearly complete regression of Tsc2-null myometrial tumors, indicating that, even without TSC2, estradiol is required to maintain tumors. Through RNAseq analysis of genes that are both estrogen and Tsc2 mediated, we identified the serine protease Neutrophil Elastase (NE) as being highly expressed in Tsc2-null uterine tumors. NE is expressed in and secreted by granulocytic neutrophils and myeloid-derived suppressor cells (MDSCs), both of which are known to be upregulated in the blood and stroma of various cancers such as breast, lung, melanoma, and prostate. NE is thought to promote pro-tumorigenic phenotypes such as proliferation, EMT, migration, invasion, and ultimately metastasis. We hypothesize that NE from infiltrating MDSCs promotes LAM tumor progression. Using flow cytometry as well as immunofluorescence, we show increased numbers of MDSCs in the blood, uterus and lungs of Tsc2-null KO mice. Immunodepleting MDSCs results in reduced tumor growth and myometrial thickness, as does blockade of MDSC recruitment to tumors using a CXCR2 antagonist. Interestingly, treatment with the NE inhibitor sivelestat phenocopies the results from MDSC depletion, leading to reduced MDSC accumulation in the tumor site, reduced tumor growth, and less myometrial thickening. Finally, using TSC2-null cell lines, we find that NE promotes aggressive tumor cell behaviors such as migration and proliferation. Thus, our data highlights the importance of MDSCs and their product NE in LAM progression, and identifies novel molecular mediators of LAM tumor growth and invasion.

Early recruitment of neutrophils from the blood to sites of tissue infection is a hallmark of innate immune responses. However, little is known about the mechanisms by which apoptotic neutrophils are cleared in infected tissues during resolution and the immunological consequences of in situ efferocytosis. Using intravital multiphoton microscopy, we show previously unrecognized motility patterns of interactions between neutrophils and tissue-resident phagocytes within the influenza-infected mouse airway. Newly infiltrated inflammatory monocytes become a chief pool of phagocytes and play a key role in the clearance of highly motile apoptotic neutrophils during the resolution phase. Apoptotic neutrophils further release epidermal growth factor and promote the differentiation of monocytes into tissue-resident antigen-presenting cells for activation of antiviral T cell effector functions. Collectively, these results suggest that the presence of in situ neutrophil resolution at the infected tissue is critical for optimal CD8+ T cell–mediated immune protection. Kim and colleagues provide new insights into the function and fate of neutrophils during influenza infection and their roles in antiviral T cell responses.

Anti-Müllerian hormone (AMH) is a member of the transforming growth factor-β superfamily and plays a crucial role in testicular and ovarian functions. In clinical practice, AMH is used as a diagnostic and/or prognostic marker in women in association with ovulation induction and in various pathophysiological conditions. Despite widespread clinical use of AMH, our mechanistic understanding of AMH actions in regulating follicular development is limited. Using a mouse model, we in this study report that in vivo AMH treatment while stalls follicular development and inhibits ovulation, also prevents follicular atresia. We further show that these AMH actions are mediated through induction of two miRNAs, miR-181a and miR-181b, which regulate various aspects of FSH signaling and follicular growth, ultimately affecting downstream gene expression and folliculogenesis. We also report that in this mouse model AMH pre-treatment prior to superovulation improves oocyte yield. These studies, therefore, offer new mechanistic insight into AMH actions in folliculogenesis and point toward potential utilization of AMH as a therapeutic agent.

Lymphangioleiomyomatosis (LAM) is a rare disease in women. Patients with LAM develop metastatic smooth-muscle cell adenomas within the lungs, resulting in reduced pulmonary function. LAM cells contain mutations in tuberous sclerosis genes (TSC1orTSC2), leading to up-regulation of mTORC1 activity and elevated proliferation. The origin of LAM cells remains unknown; however, inactivation ofTsc2gene in the mouse uterus resulted in myometrial tumors exhibiting LAM features, and approximately 50% of animals developed metastatic myometrial lung tumors. This suggests that LAM tumors might originate from the uterine myometrium, possibly explaining the overwhelming prevalence of LAM in female. Here, we demonstrate that mouseTsc2-null myometrial tumors exhibit nearly all the features of LAM, including mTORC1/S6K activation, as well as expression of melanocytic markers and matrix metalloproteinases (MMPs). Estrogen ablation reduces S6K signaling and results inTsc2-null myometrial tumor regression. Thus, even without TSC2, estradiol is required to maintain tumors and mTORC1/S6K signaling. Additionally, we find that MMP-2 and -9, as well as neutrophil elastase (NE), are overexpressed inTsc2-null myometrial tumors in an estrogen-dependent fashion.In vivofluorescent imaging using MMP- or NE-sensitive optical biomarkers confirms that protease activity is specific to myometrial tumors. Similar to LAM cells, uterineTsc2-null myometrial cells also overexpress melanocytic markers in an estrogen-dependent fashion. Finally, we identify glycoprotein NMB (GPNMB) as a melanocytic marker up-regulated inTsc2-null mouse uteri and human LAM samples. Our data highlight the potential importance of estradiol in LAM cells, suggesting that anti-estrogen therapy may be a treatment modality. Furthermore, proteases and GPNMB might be useful LAM biomarkers.

Obesity is considered detrimental to women's reproductive health. Although most of the attention has been focused on the effects of obesity on hypothalamic function, studies suggest a multifactorial impact. In fact, obesity is associated with reduced fecundity even in women with regular cycles, indicating that there may be local ovarian effects modulating fertility. Here we describe a novel mechanism for leptin actions directly in the ovary that may account for some of the negative effects of obesity on ovarian function. We find that normal cycling, obese, hyperleptinemic mice fed with a high-fat diet are subfertile and ovulate fewer oocytes compared with animals fed with a normal diet. Importantly, we show that leptin induces expression of the neuropeptide cocaine- and amphetamine-regulated transcript (CART) in the granulosa cells (GCs) of ovarian follicles both in vitro and in vivo. CART then negatively affects intracellular cAMP levels, MAPK signaling, and aromatase mRNA expression, which leads to lower estradiol synthesis in GCs and altered ovarian folliculogenesis. Finally, in human samples from patients undergoing in vitro fertilization, we show a significant positive correlation between patient body mass index, CART mRNA expression in GCs, and CART peptide levels in follicular fluid. These observations suggest that, under obese conditions, CART acts as a local mediator of leptin in the ovary to cause ovarian dysfunction and reduced fertility.

Efficient recruitment and correct positioning of T cells within infected peripheral tissues are crucial for pathogen clearance. Using a dual-chemokine reporter (Groom et Al. Immunity, 2012) mouse and intra-vital imaging, we identified a preferred tissue entry and accumulation site for effector Th1 cells, defined by clusters of CXCL9 and CXCL10 expressing hematopoietic cells, enriched with MHC-class-II+ cells, in restricted perivascular spaces within the inflamed dermis. Unbiased computational analysis of motility dynamics of antigen-specific Th1 cells within these clusters, revealed cell confinement characteristics. CXCR3 on Th1 cells and presence of cognate antigen were critical for persistence within clusters and T cell activation. Newly recruited Th1 cells strongly amplified CXCL9 and CXCL10 expression, mainly within skin monocyte-derived dendritic cells, in an antigen- and interferon-gamma-dependent manner, resulting in augmented chemokine clusters. Our data suggest a CXCR3-mediated mechanism of intrinsic amplification loop for optimal Th1 cell recruitment, positioning and activation in restricted antigen presentation sites within the inflamed skin.

For many decades, elevated androgens in women have been associated with poor reproductive health. However, recent studies have shown that androgens play a crucial role in women's fertility. The following review provides an overall perspective about how androgens and androgen receptor-mediated actions regulate normal follicular development, as well as discuss emerging concepts, latest perceptions, and controversies regarding androgen actions and signaling in the ovary.

Although androgen excess is considered detrimental to women's health and fertility, global and ovarian granulosa cell-specific androgen-receptor (AR) knockout mouse models have been used to show that androgen actions through ARs are actually necessary for normal ovarian function and female fertility. Here we describe two AR-mediated pathways in granulosa cells that regulate ovarian follicular development and therefore female fertility. First, we show that androgens attenuate follicular atresia through nuclear and extranuclear signaling pathways by enhancing expression of the microRNA (miR) miR-125b, which in turn suppresses proapoptotic protein expression. Second, we demonstrate that, independent of transcription, androgens enhance follicle-stimulating hormone (FSH) receptor expression, which then augments FSH-mediated follicle growth and development. Interestingly, we find that the scaffold molecule paxillin regulates both processes, making it a critical regulator of AR actions in the ovary. Finally, we report that low doses of exogenous androgens enhance gonadotropin-induced ovulation in mice, further demonstrating the critical role that androgens play in follicular development and fertility. These data may explain reported positive effects of androgens on ovulation rates in women with diminished ovarian reserve. Furthermore, this study demonstrates mechanisms that might contribute to the unregulated follicle growth seen in diseases of excess androgens such as polycystic ovary syndrome.

T-helper (Th) cell differentiation drives specialized gene programs that dictate effector T cell function at sites of infection. Here we have shown Th cell differentiation also imposes discrete motility gene programs that shape Th1 and Th2 cell navigation of the inflamed dermis. Th1 cells scanned a smaller tissue area in a G-protein coupled receptor (GPCR) and chemokine-dependent fashion while Th2 cells scanned a larger tissue area independent of GPCR signals. Differential chemokine reliance for interstitial migration was linked to STAT6 transcription factor-dependent programming of integrin α(V)β(3) expression: Th2 cell differentiation led to high α(V)β(3) expression relative to Th1 cells. Th1 and Th2 cell modes of motility could be switched simply by manipulating the amount of α(V)β(3) on the cell surface. Deviating motility modes from those established during differentiation impaired effector function. Thus, programmed expression of α(V)β(3) tunes effector T cell reliance on environmental cues for optimal exploration of inflamed tissues.

Intracellular pathogen clearance is dependent on successful T cell mediated immunity. Th1 cells must localize to the site of infection to be activated into IFN-γ producing effector T cells. How Th1 cells find and interact with antigen presenting cells (APC) in infected tissues is poorly understood. Preliminary data using intravital imaging and the REX3 mouse, in which CXCL9 and CXCL10 producing cells express red and blue fluorescent proteins (RFP/BFP) respectively, suggest that CXCL9/10-producing cells form dense perivascular clusters that attract and arrest CXCR3+ Th1 cells. We hypothesize that these CXCL10+ cell clusters are enriched for APC that optimize Th1 cell activation and IFN-γ production. To determine the phenotype of the immune cells within the chemokine-producing clusters we crossed the REX3 mouse with a photoactivatable green fluorescent protein (PA-GFP) mouse. This will enable selective labeling of cells within chemokine-rich clusters prior to flow cytometry. Using precise multiphoton delivery of 830 nm light, we have successfully photoactivated the CXCL10+ clustered regions in the inflamed ear and have developed a flow cytometry panel to distinguish between macrophages, cDCs, and monocyte-derived cells that may be positioned within or outside these chemokine-clusters. Additionally, we will determine if these cell clusters enhance Th1 function by sorting photoactivated (in clusters) and non-photoactivated (outside clusters) Th1 cells from the inflamed ear and analyzing IFN-γ expression. Understanding how Th1 cells are positioned and activated at sites of inflammation will allow the field to boost responses to infectious agents and therapeutically attenuate Th1 responses in autoimmunity.

Lymphangioleiomyomatosis (LAM) is a devastating rare lung disease affecting primarily childbearing age women in which tumors consisting of abnormal smooth-muscle-like cells grow within the lungs and progressively lead to loss of pulmonary function. LAM cells metastasize to the lungs, predominantly through the lymphatics; however, the source of the LAM cell is still unknown. LAM cells contain inactivating mutations in genes encoding tuberous sclerosis 1 or 2, proteins that normally limit cell growth through suppression of mammalian target of rapamycin complex 1. As of today, sirolimus (an mammalian target of rapamycin complex 1 inhibitor) is the only treatment, available for LAM patients that is approved by the Food and Drug Administration; however, this drug and others in its class provide stabilization but not remission of LAM. One of the biggest problems in treating LAM is that both the origin of the LAM cells and the mechanism of the sexual dimorphism in LAM are still not understood. LAM cells express estrogen and progesterone receptors, and lung function declines during periods of high circulating estrogen levels. Moreover, numerous basic research studies find that estrogen is a key driving force in LAM cell proliferation, migration, and metastasis. In this review, we highlight recent insights regarding the role of steroid hormones in LAM and discuss possible explanations for the profound female sexual dimorphism of LAM.

To exit blood vessels, lumenally adhered monocytes and neutrophils crawl towards specific locations that support transmigration. Using intravital confocal microscopy of anesthetized mouse cremaster muscle we separately examined the intralumenal crawling of blood monocytes versus neutrophils and determined that CD11a/CD18 (LFA-1) versus CD11b/CD18 (Mac-1) mediated crawling accounted for these differences. In unstimulated venules 58.2±6.1% of all monocytes, but only 13.6±0.9% of all neutrophils were found adhered; TNFα treatment increased the number of adhered monocytes and neutrophils 2- and 3-fold respectively. The majority (~80%) of all adhered leukocytes exhibited intralumenal crawling under both unstimulated and activated conditions. Function blocking antibodies directed against Mac-1 but not LFA-1 significantly attenuated neutrophil crawling in unstimulated and TNFα activated venules, indicating that under both conditions neutrophil crawling is Mac-1 dependent. In contrast, treatment with combinations of Mac-1 and LFA-1 blocking antibodies reveled that both LFA-1 and Mac-1 contribute to monocyte crawling, however the dominant role in mediating monocyte crawling changes from LFA-1 in unstimulated venules to Mac-1 upon inflammation, likely due to increased expression of Mac-1 under these conditions. We show that LFA-1 mediated crawling is for 2–3 fold longer distances than that crawling mediated by Mac-1. Consistent with the role of Mac-1 in crawling, Mac-1 block (compared to LFA-1) was also significantly more efficient in blocking TNFα induced extravasation of both monocytes and neutrophils in both cremaster tissue and the peritoneal cavity. Thus, mechanisms underlying leukocyte crawling are important in regulating the inflammatory responses by regulating the numbers of transmigrated leukocytes.

Lymphangioleiomyomatosis (LAM) is a rare disease characterized by proliferation of abnormal smooth-muscle cells in the lungs, leading to functional loss and sometimes lung transplantation. Although the origin of LAM cells is unknown, several features of LAM provide clues. First, LAM cells contain inactivating mutations in genes encoding Tsc1 or Tsc2, proteins that limit mTORC1 activity. Second, LAM tumors recur after lung transplantation, suggesting a metastatic pathogenesis. Third, LAM is found almost exclusively in women. Finally, LAM shares features with uterine leiomyomas, benign tumors of myometrial cells. From these observations, we proposed that LAM cells might originate from uterine leiomyomas containing Tsc mutations. To test our hypothesis, and to develop mouse models for leiomyoma and LAM, we targeted Tsc2 deletion primarily in uterine cells. In fact, nearly 100% of uteri from uterine-specific Tsc2 knockout mice developed myometrial proliferation and uterine leiomyomas by 12 and 24 weeks, respectively. Myometrial proliferation and mTORC1/S6 activity were abrogated by the mTORC1 inhibitor rapamycin or by elimination of sex steroid production through ovariectomy or aromatase inhibition. In ovariectomized Tsc2 null mice, mTORC1/S6 activity and myometrial growth were restored by estrogen but not progesterone. Thus, even without Tsc2, estrogen appears to be required for myometrial mTORC1/S6 signaling and proliferation. Finally, we found Tsc2 null myometrial tumors in lungs of older Tsc2 uterine-specific knockout females, suggesting that lung LAM-like myometrial lesions may indeed originate from the uterus. This mouse model may improve our understanding of LAM and leiomyomas and might lead to novel therapeutic strategies for both diseases.

Previous work has demonstrated that cross talk between G protein-coupled LH receptors and epidermal growth factor receptors (EGFR) is essential for LH-induced steroid production in ovarian follicles and testicular Leydig cells. Here we demonstrate that G protein-coupled receptor (GPCR)/EGFR cross talk is also required for ACTH-induced steroidogenesis in Y1 adrenal cells. Moreover, we confirm that the signaling pathway from GPCR to Erk activation is conserved in all three steroidogenic tissues. ACTH or LH induces Gα(s), resulting in elevated cAMP and protein kinase A activation. cAMP/protein kinase A then triggers EGFR trans-activation, which promotes Erk signaling and subsequent steroidogenesis. Interestingly, although EGFR trans-activation is conserved in all three tissues, the specific mechanisms regulating this receptor cross talk differ. ACTH and LH trigger matrix metalloproteinase (MMP)-mediated release of EGFR ligands in adrenal and gonadal cells, respectively. However, this extracellular, ligand-dependent EGFR transactivation is required only for LH-induced steroidogenesis in ovarian follicles, reflecting the unique requirement of cell-cell cross talk for ovarian steroid production. Furthermore, MMP2 and MMP9 appear to regulate LH-induced steroidogenesis in mouse ovarian follicles, because a specific MMP2/9 inhibitor as well as the MMP2/9 inhibitor doxycycline suppress LH-induced follicular steroid production in vitro. Notably, although EGFR or MMP inhibition minimally affects estrous cycling in female mice, they attenuate ovarian steroidogenesis in response to LHR overstimulation in vivo. These results may have implications with regard to EGFR inhibitor use in various cancers as well as in polycystic ovarian syndrome, where excess LH-driven ovarian androgen production might be controlled by MMP2/9 inhibition.

Steroids are key factors in a myriad of mammalian biological systems, including the brain, kidney, heart, bones, and gonads. While alternative potential steroid receptors have been described, the majority of biologically relevant steroid responses appear to be mediated by classical steroid receptors that are located in all parts of the cell, from the plasma membrane to the nucleus. Interestingly, these classical steroid receptors modulate different signals depending upon their location. For example, receptors in the plasma membrane interact with membrane signaling molecules, including G proteins and kinases. In contrast, receptors in the nucleus interact with nuclear signaling molecules, including transcriptional co-regulators. These extranuclear and intranuclear signals function together in an integrated fashion to regulate important biological functions. While most studies on extranuclear steroid signaling have focused on estrogens, recent work has demonstrated that nongenomic androgen signaling is equally important and that these two steroids modulate similar signaling pathways. In fact, by taking advantage of a simple model system whereby a physiologically relevant androgen-mediated process is regulated completely independent of transcription (Xenopus laevis oocyte maturation), many novel and conserved concepts in nongenomic steroid signaling have been uncovered and characterized.