Ovarian steroid withdrawal results in GABAA receptor upregulation in the photoperiodic neuroendocrine pathways of the turkey hen.

The mechanism(s) underlying photorefractoriness in temperate zone seasonally breeding birds remains undetermined. Our recent findings reveal a link between the upregulation of GABAA receptors (GABAARs) in the premammillary nucleus (PMM) and the state of photorefractoriness. Gonadal steroid levels fluctuate during the breeding season; increasing after gonadal recrudescence and declining sharply once gonadal regression begins. Here, we examined the effect of gonadal steroid withdrawal on the expression of GABAARs in the turkey PMM. Exogenous ovarian steroids were administered and then withdrawn from turkey hens to mimic the decline of ovarian steroids levels at the end of a breeding season. The upregulation of GABAAR α3, α4, δ, π, and γ2-subunits was observed in the PMM of the steroid withdrawal group when compared to the non-steroid treatment group. The level of tyrosine hydroxylase, photopigment melanopsin, and circadian clock genes in the PMM of the steroid withdrawal group resembled the levels observed in the natural photorefractory hens and were significantly lower than those of the short-day light stimulated group. A reduction in gonadotropin-releasing hormone-I mRNA expressed within the nucleus commissurae pallii was also observed in hens undergoing steroid withdrawal. These results suggest that the natural decline in circulating ovarian steroid levels may modulate the GABAergic system in the PMM through the upregulation of GABAA receptors. This, in turn, could diminish the reproductive neuroendocrine responses to light and favor a condition resembling the state of photorefractoriness.

GABAergic Neurotransmission in the Premammillary Nucleus of the Turkey Hypothalamus Regulates Reproductive Seasonality and the Onset of Photorefractoriness.

BACKGROUND/AIMS: Photoperiod is a major environmental cue in temperate-zone birds which synchronizes breeding with the time of year that offers the optimal environment for offspring survival. Despite continued long photoperiods, these birds eventually become refractory to the stimulating photoperiod and their reproductive systems regress. In this study, we characterized the role of γ-aminobutyric acid (GABA)ergic neurotransmission in modulating the response of the premammillary nucleus (PMM) to a gonad stimulatory photoperiod and the onset of photorefractoriness. METHODS AND RESULTS: Bilateral ablation of the PMM blocked the light-induced neuroendocrine response from occurring in photosensitive turkeys. Microarray analyses revealed an increase in GABAergic activity in the PMM of photorefractory birds as opposed to photosensitive ones, and this enhanced GABAergic activity appeared to inhibit the photoperiodic signal. Additionally, GABAA and GABAB receptors were expressed by dopamine-melatonin neurons in the PMM, and the administration of the GABA receptor agonist baclofen blocked the photoperiodic reproductive neuroendocrine responses. CONCLUSIONS: Consistent with the present findings, we propose that the long-sought-after mechanism underlying photorefractoriness is linked to the inhibitory actions of GABA. We suggest that (1) GABAergic interference with photoperiodic entrainment in the PMM initiates the photorefractory state and terminates the annual breeding season in temperate-zone birds, and (2) the PMM is a site of photoreception and photorefractoriness that controls the initiation and termination of avian reproductive seasonality.

Enhanced GABAergic inhibition in the premammillary nucleus of photorefractory turkey hens via GABAA receptor upregulation.

The premammillary nucleus (PMM) of the turkey mediobasal hypothalamus, where dopamine-melatonin (DA-Mel) neurons are localized, is a site for photoreception and photoperiodic time measurement, which is essential for the initiation of avian reproductive seasonality. In addition, this area could also be responsible for the onset and maintenance of photorefractoriness at the end of the breeding season due to the enhanced inhibitory effect of γ-aminobutyric acid (GABA). GABA is an inhibitory neurotransmitter in the central nervous system which interferes with the photosexual response in the turkey, a seasonally breeding bird. Here, we further characterized the GABAA receptor subunits in the PMM DA-Mel neurons related to reproductive seasonality and the onset of photorefractoriness. GABAA receptor subunits and GABA synthesis enzymes in the PMM of photosensitive and photorefractory turkey hens were identified using real-time qRT-PCR. The upregulation of GABAA receptor α1-3, ß2-3, γ1-3, ρ1-3, δ, and θ mRNA expression were observed in the PMM of photorefractory birds when compared to those of photosensitive ones while there is no change observed in the GABA synthesis enzymes, glutamate decarboxylase 1 and 2. Those upregulated GABAA receptor subunits were further examined using immunohistochemical staining and they appeared to be co-localized within the PMM DA-Mel neurons. The upregulation of GABAA receptor subunits observed in the PMM of photorefractory birds coincides with a lack of responsiveness to a light stimulus provided during the photosensitive phase. This is supported by the absence of c-fos induction and TH upregulation in the PMM and a subsequence inhibition of c-fos and GnRH-I expression in the nucleus commissurae pallii. The augmented GABAA receptor subunits expression may mediate an enhancement of inhibitory GABAergic neurotransmission and the subsequent interference with the photosexual response. This could contribute to the state of photorefractoriness and the termination of breeding activities in the turkey, a temperate zone bird.

Photoreceptive oscillators within neurons of the premammillary nucleus (PMM) and seasonal reproduction in temperate zone birds.

The pathway for light transmission regulating the reproductive neuroendocrine system in temperate zone birds remains elusive. Based on the evidence provided from our studies with female turkeys, it is suggested that the circadian clock regulating reproductive seasonality is located in putatively photosensitive dopamine-melatonin (DA-MEL) neurons residing in the premammillary nucleus (PMM) of the caudal hypothalamus. Melanopsin is expressed by these neurons; a known photopigment which mediates light information pertaining to the entrainment of the clock. Exposure to a gonad stimulatory photoperiod enhances the activity of the DAergic system within DA-MEL neurons. DAergic activity encoding the light information is transmitted to the pars tuberalis, where thyroid-stimulating hormone, beta (TSHß) cells reside, and induces the release of TSH. TSH stimulates tanycytes lining the base of the third ventricle and activates type 2 deiodinase in the ependymal which enhances triiodothyronine (T3) synthesis. T3 facilitates the release of gonadotropin-releasing hormone-I which stimulates luteinizing hormone/follicle stimulating hormone release and gonad recrudescence. These data taken together with the findings that clock genes are rhythmically expressed in the PMM where DA-MEL neurons are localized imply that endogenous oscillators containing photoreceptors within DA-MEL neurons are important in regulating the DA and MEL rhythms that drive the circadian cycle controlling seasonal reproduction.

Navigating a plethora of progesterone receptors: Comments on the safety/risk of progesterone supplementation in women with a history of breast cancer or at high-risk for developing breast cancer.

Progesterone and progestin agonists are potent steroid hormones. There are at least three major types of progesterone receptor (PR) families that interact with and respond to progesterone or progestin ligands. These receptors include ligand-activated transcription factor isoforms (PR-A and PR-B) encoded by the PGR gene, often termed classical or nuclear progesterone receptor (nPR), membrane-spanning progesterone receptor membrane component proteins known as PGRMC1/2, and a large family of progestin/adipoQreceptors or PAQRs (also called membrane PRs or mPRs). Cross-talk between mPRs and nPRs has also been reported. The complexity of progesterone actions via a plethora of diverse receptors warrants careful consideration of the clinical applications of progesterone, which primarily include birth control formulations in young women and hormone replacement therapy following menopause. Herein, we focus on the benefits and risk of progesterone/progestin supplementation. We conclude that progesterone-only supplementation is considered safe for most reproductive-age women. However, women who currently have ER + breast cancer or have had such cancer in the past should not take sex hormones, including progesterone. Women at high-risk for developing breast or ovarian cancer, either due to their family history or known genetic factors (such as BRCA1/2 mutation) or hormonal conditions, should avoid exogenous sex hormones and proceed with caution when considering using natural hormones to mitigate menopausal symptoms and/or improve quality of life after menopause. These individuals are urged to consult with a qualified OB-GYN physician to thoroughly assess the risks and benefits of sex hormone supplementation. As new insights into the homeostatic roles and specificity of highly integrated rapid signaling and nPR actions are revealed, we are hopeful that the benefits of using progesterone use may be fully realized without an increased risk of women's cancer.

Reevaluating the Role of Progesterone in Ovarian Cancer: Is Progesterone Always Protective?

Ovarian cancer (OC) represents a collection of rare but lethal gynecologic cancers where the difficulty of early detection due to an often-subtle range of abdominal symptoms contributes to high fatality rates. With the exception of BRCA1/2 mutation carriers, OC most often manifests as a post-menopausal disease, a time in which the ovaries regress and circulating reproductive hormones diminish. Progesterone is thought to be a "protective" hormone that counters the proliferative actions of estrogen, as can be observed in the uterus or breast. Like other steroid hormone receptor family members, the transcriptional activity of the nuclear progesterone receptor (nPR) may be ligand dependent or independent and is fully integrated with other ubiquitous cell signaling pathways often altered in cancers. Emerging evidence in OC models challenges the singular protective role of progesterone/nPR. Herein, we integrate the historical perspective of progesterone on OC development and progression with exciting new research findings and critical interpretations to help paint a broader picture of the role of progesterone and nPR signaling in OC. We hope to alleviate some of the controversy around the role of progesterone and give insight into the importance of nPR actions in disease progression. A new perspective on the role of progesterone and nPR signaling integration will raise awareness to the complexity of nPRs and nPR-driven gene regulation in OC, help to reveal novel biomarkers, and lend critical knowledge for the development of better therapeutic strategies.

Progesterone Receptors Promote Quiescence and Ovarian Cancer Cell Phenotypes via DREAM in p53-Mutant Fallopian Tube Models.

CONTEXT: The ability of ovarian steroids to modify ovarian cancer (OC) risk remains controversial. Progesterone is considered to be protective; recent studies indicate no effect or enhanced OC risk. Knowledge of progesterone receptor (PR) signaling during altered physiology that typifies OC development is limited. OBJECTIVE: This study defines PR-driven oncogenic signaling mechanisms in p53-mutant human fallopian tube epithelia (hFTE), a precursor of the most aggressive OC subtype. METHODS: PR expression in clinical samples of serous tubal intraepithelial carcinoma (STIC) lesions and high-grade serous OC (HGSC) tumors was analyzed. Novel PR-A and PR-B isoform-expressing hFTE models were characterized for gene expression and cell cycle progression, emboli formation, and invasion. PR regulation of the DREAM quiescence complex and DYRK1 kinases was established. RESULTS: STICs and HGSC express abundant activated phospho-PR. Progestin promoted reversible hFTE cell cycle arrest, spheroid formation, and invasion. RNAseq/biochemical studies revealed potent ligand-independent/-dependent PR actions, progestin-induced regulation of the DREAM quiescence complex, and cell cycle target genes through enhanced complex formation and chromatin recruitment. Disruption of DREAM/DYRK1s by pharmacological inhibition, HPV E6/E7 expression, or DYRK1A/B depletion blocked progestin-induced cell arrest and attenuated PR-driven gene expression and associated OC phenotypes. CONCLUSION: Activated PRs support quiescence and pro-survival/pro-dissemination cell behaviors that may contribute to early HGSC progression. Our data support an alternative perspective on the tenet that progesterone always confers protection against OC. STICs can reside undetected for decades prior to invasive disease; our studies reveal clinical opportunities to prevent the ultimate development of HGSC by targeting PRs, DREAM, and/or DYRKs.

Secretion of MCP-1 and other paracrine factors in a novel tumor-bone coculture model.

BACKGROUND: The bone-tumor microenvironment encompasses unique interactions between the normal cells of the bone and marrow cavity and the malignant cells from a primary or metastasized cancer. A multitude of paracrine factors within this microenvironment such as the growth factor, TGF-beta, and the chemokine, MCP-1, are secreted by many of these cell types. These factors can act in concert to modulate normal and malignant cell proliferation, malignant cell migration and invasion and, often, mediate bone cancer pain. Although many valuable in vitro and in vivo models exist, identifying the relevant paracrine factors and deciphering their interactions is still a challenge. The aim of our study is to test an ex vivo coculture model that will allow monitoring of the expression, release and regulation of paracrine factors during interactions of an intact femur explant and tumor cells. METHODS: Intact or marrow-depleted neonatal mouse femurs and select murine and human sarcoma or carcinoma cell lines were incubated singly or in coculture in specialized well plates. Viability of the bone and cells was determined by immunohistochemical stains, microscopy and marrow cytopreps. Secretion and mRNA expression of paracrine factors was quantitated by ELISA and real-time RT-PCR. RESULTS: Compartments of the bone were optimally viable for up to 48 h in culture and tumor cells for up to 4 days. Bone was the major contributor of TGF-beta and MMP2 whereas both bone and sarcoma cells secreted the chemokine MCP-1 in cocultures. Synergistic interaction between the femur and sarcoma resulted in enhanced MCP-1 secretion and expression in cocultures and was dependent on the presence of the hematopoietic component of the bone as well as other bone cells. In contrast, coculturing with breast carcinoma cells resulted in reduction of TGF-beta and MCP-1 secretion from the bone. CONCLUSION: These studies illustrate the feasibility of this model to examine paracrine interactions between intact bone and tumor cells. Further study of unique regulation of MCP-1 secretion and signaling between these cell types in different types of cancer will be possible using this simulated microenvironment.

Reciprocal regulation of extracellular signal regulated kinase 1/2 and mitogen activated protein kinase phosphatase-3.

Mitogen activated protein kinase phosphatase-3 (MKP-3) is a putative tumor suppressor. When transiently overexpressed, MKP-3 dephosphorylates and inactivates extracellular signal regulated kinase (ERK) 1/2. Little is known about the roles of endogenous MKP-3, however. We previously showed that MKP-3 is upregulated in cell lines that express oncogenic Ras. Here we tested the roles of endogenous MKP-3 in modulating ERK1/2 under conditions of chronic stimulation of the Ras/Raf/MEK1/2/ERK1/2 pathway by expression of oncogenic Ras. We used two cell lines: H-ras MCF10A, breast epithelial cells engineered to express H-Ras, and DLD-1, colon cancer cells that express endogenous Ki-Ras. First, we found that MKP-3 acts in a negative feedback loop to suppress basal ERK1/2 when oncogenic Ras stimulates the Ras/Raf/MEK1/2/ERK1/2 cascade. ERK1/2 was required to maintain elevated MKP-3, indicative of a negative feedback loop. Accordingly, knockdown of MKP-3, via siRNA, increased ERK1/2 phosphorylation. Second, by using siRNA, we found that MKP-3 helps establish the sensitivity of ERK1/2 to extracellular activators by limiting the duration of ERK1/2 phosphorylation. Third, we found that the regulation of ERK1/2 by MKP-3 is countered by the complex regulation of MKP-3 by ERK1/2. Potent ERK1/2 activators stimulated the loss of MKP-3 within 30 min due to an ERK1/2-dependent decrease in MKP-3 protein stability. MKP-3 levels recovered within 120 min due to ERK1/2-dependent resynthesis. Preventing MKP-3 resynthesis, via siRNA, prolonged ERK1/2 phosphorylation. Altogether, these results suggest that under the pressure of oncogenic Ras expression, MKP-3 reins in ERK1/2 by serving in ERK1/2-dependent negative feedback pathways.

Progesterone receptors (PR) mediate STAT actions: PR and prolactin receptor signaling crosstalk in breast cancer models.

Estrogen is the major mitogenic stimulus of mammary gland development during puberty wherein ER signaling acts to induce abundant PR expression. PR signaling, in contrast, is the primary driver of mammary epithelial cell proliferation in adulthood. The high circulating levels of progesterone during pregnancy signal through PR, inducing expression of the prolactin receptor (PRLR). Cooperation between PR and prolactin (PRL) signaling, via regulation of downstream components in the PRL signaling pathway including JAKs and STATs, facilitates the alveolar morphogenesis observed during pregnancy. Indeed, these pathways are fully integrated via activation of shared signaling pathways (i.e. JAKs, MAPKs) as well as by the convergence of PRs and STATs at target genes relevant to both mammary gland biology and breast cancer progression (i.e. proliferation, stem cell outgrowth, tissue cell type heterogeneity). Thus, rather than a single mediator such as ER, transcription factor cascades (ER>PR>STATs) are responsible for rapid proliferative and developmental programming in the normal mammary gland. It is not surprising that these same mediators typify uncontrolled proliferation in a majority of breast cancers, where ER and PR are most often co-expressed and may cooperate to drive malignant tumor progression. This review will primarily focus on the integration of PR and PRL signaling in breast cancer models and the importance of this cross-talk in cancer progression in the context of mammographic density. Components of these PR/PRL signaling pathways could offer alternative drug targets and logical complements to anti-ER or anti-estrogen-based endocrine therapies.

Progesterone action in breast, uterine, and ovarian cancers.

Progesterone and progesterone receptors (PRs) are essential for the development and cyclical regulation of hormone-responsive tissues including the breast and reproductive tract. Altered functions of PR isoforms contribute to the pathogenesis of tumors that arise in these tissues. In the breast, progesterone acts in concert with estrogen to promote proliferative and pro-survival gene programs. In sharp contrast, progesterone inhibits estrogen-driven growth in the uterus and protects the ovary from neoplastic transformation. Progesterone-dependent actions and associated biology in diverse tissues and tumors are mediated by two PR isoforms, PR-A and PR-B. These isoforms are subject to altered transcriptional activity or expression levels, differential crosstalk with growth factor signaling pathways, and distinct post-translational modifications and cofactor-binding partners. Herein, we summarize and discuss the recent literature focused on progesterone and PR isoform-specific actions in breast, uterine, and ovarian cancers. Understanding the complexity of context-dependent PR actions in these tissues is critical to developing new models that will allow us to advance our knowledge base with the goal of revealing novel and efficacious therapeutic regimens for these hormone-responsive diseases.

Tyrosine phosphatases as regulators of skeletal development and metabolism.

The protein tyrosine kinases (PTK) and the protein tyrosine phosphatases (PTPs) are enzymes which play an integral role in tyrosine phosphorylation-dependent signaling cascades. By catalyzing the phosphorylation and dephosphorylation of cellular proteins, these enzymes direct the steady-state levels of specific phosphoproteins and ultimately dictate the functional state of all cells. The importance of this type of signaling in the skeleton is accepted but poorly understood. The contribution of the PTKs to signaling events in bone has been well studied but, in contrast, the regulation by PTPs is poorly defined. The recent identification of 107 genes within the human genome which encode members of the PTP superfamily emphasizes the need to consider the importance of these proteins in skeletal tissue. In this prospective, we will summarize the present state of our knowledge regarding the function of this enzyme superfamily, illustrating its relevance to the development and maintenance of the skeleton and highlighting future directions that should improve our understanding of these critical signaling molecules.

Mitogen-activated protein kinase phosphatase-3 is a tumor promoter target in initiated cells that express oncogenic Ras.

We have capitalized on the unique properties of the skin tumor promoter palytoxin, which does not activate protein kinase C, to investigate alternative mechanisms by which major signaling molecules can be modulated during carcinogenesis. We report here that palytoxin activates extracellular signal-regulated kinase (ERK) through a novel mechanism that involves inactivation of an ERK phosphatase in keratinocytes derived from initiated mouse skin (308 cells). Use of U0126 revealed that palytoxin requires the ERK kinase MEK to stimulate ERK activity, although palytoxin did not activate MEK. We found that 308 keratinocytes highly express mitogen-activated protein kinase phosphatase-3 (MKP-3), which selectively inactivates ERK. Palytoxin induced the loss of MKP-3 in a manner that corresponded to increased ERK phosphorylation. Complementary studies showed that sustained expression of exogenous MKP-3 inhibited palytoxin-stimulated ERK activation. As is characteristic of initiated keratinocytes, 308 cells express activated H-Ras. To investigate whether expression of oncogenic Ras is key to palytoxin-stimulated ERK activation, we determined how palytoxin affected ERK and MKP-3 in MCF10A human breast epithelial cells and in H-ras MCF10A cells, which stably express activated H-Ras. Palytoxin did not affect ERK activity in MCF10A cells, which had no detectable MKP-3. Like 308 cells, H-ras MCF10A cells highly express MKP-3. Strikingly, palytoxin stimulated ERK activity and induced a corresponding loss of MKP-3 in H-ras MCF10A cells. These studies indicate that in initiated cells palytoxin unleashes ERK activity by down-regulating MKP-3, an ERK inhibitor, and further suggest that MKP-3 may be a vulnerable target in cells that express oncogenic Ras.

Transcriptional activation of the tyrosine phosphatase gene, OST-PTP, during osteoblast differentiation.

Protein tyrosine phosphatases (PTPs) are critical regulators of cellular phosphorylation functioning in processes such as cell growth, differentiation, and adhesion. Osteotesticular PTP (OST) is the only characterized member of this superfamily whose expression is regulated in osteoblasts and critical for their in vitro differentiation. Such evidence would suggest that this molecule is a key modulator of signaling events during osteogenesis, yet little is known about its genetic regulation. In an effort to examine the molecular mechanisms involved in the cellular regulation of OST, we have characterized its expression in MC3T3 osteoblasts during differentiation. Northern analysis revealed that murine OST mRNA is dramatically regulated during the preosteoblast to osteoblast progression, with predominant expression in differentiated and early mineralizing osteoblasts. This expression pattern is unique to this phosphatase since, in comparison, the structurally similar receptor PTP, LAR, and the intracellular PTP1B show little change during differentiation. Cell density contributes to this upregulated expression as confluent cultures display an increase in OST transcripts within 4 h post-plating. Transient transfection of the OST promoter in differentiating MC3T3 results in a significant increase in transcriptional activation from day 0 to day 5 of differentiation, similar in timing and intensity to the observed upregulation of the endogenous gene. This activation appears to be specific to osteoblasts, since progression to a myoblast phenotype results in no change in reporter gene activity. Culturing these preosteoblast cells in the absence of critical co-factors results in an inhibition of differentiation and leads to a delayed induction of OST transcripts as well as the attenuation of transcriptional activation. These results show that the murine OST gene is regulated at the transcriptional level in an osteoblast-specific, differentiation-dependent manner during the differentiation of MC3T3 osteoblasts. Future studies will help determine the essential regulatory elements within the OST-PTP promoter and the critical signaling pathways important in this regulation.

The tyrosine phosphatase, OST-PTP, is expressed in mesenchymal progenitor cells early during skeletogenesis in the mouse.

Osteotesticular protein tyrosine phosphatase (OST-PTP; OST), is a signaling molecule which catalyzes the removal of phosphates from tyrosine residues. It is known to be highly regulated in bone cells and has been shown to be important for the in vitro progression from a preosteoblast to a mature, mineralizing cell. However, the in vivo expression of this phosphatase during skeletogenesis has not been examined. Using Northern analysis and in situ hybridization (ISH), we have observed that this gene is strongly expressed early during the formation of the mouse skeleton. By 12.5 days postcoitum (dpc), expression of OST mRNA transcripts increases and is localized within the mesenchyme of craniofacial bones, ribs, limbs, and Meckel's cartilage. Following initiation of chondrogenesis, OST mRNA becomes restricted to the perichondrium of all endochondral elements. With ossification, this gene is also expressed by cells, presumably osteoblasts, at the chondro-osseous border and along cortical and trabecular bone surfaces. Unlike other bone markers examined such as Osterix and type II collagen, OST transcripts do not appear to be expressed by chondrocytes of epiphyseal cartilage or by non-hypertrophic or hypertrophic chondrocytes. Because the temporal expression patterns of OST and Runx2 were similar suggesting a potential interrelationship in bone regulation and function, OST expression was examined in transgenic mice lacking a functional Runx2/Cbfal protein (Runx2/Cbfal delta C (deltaC)) and possessing a cartilaginous skeleton. Interestingly, the OST gene was expressed with localization similar in wild-type, homozygous, and heterozygous embryos. These studies suggest that the expression of the OST gene may be important during skeletogenesis, potentially from commitment of mesenchymal cells to the ossification of new bones. Early in embryogenesis, regulation of OST expression may be independent of Runx2/Cbfa1.

Tumor promoter-induced MMP-13 gene expression in a model of initiated epidermis.

In mouse epidermis in vivo, the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increases gene expression of matrix metalloproteinase-13 (MMP-13), an enzyme implicated in carcinogenesis. Here we used a keratinocyte cell line (308) derived from initiated mouse skin to investigate TPA-induced MMP-13 gene expression. Use of a pharmacological inhibitor (U0126) demonstrated that extracellular signal regulated kinase (ERK) plays a major role in TPA-induced MMP-13 gene expression. The 5'-flanking sequences of the MMP-13 gene contain binding sites for activator protein-1 (AP-1) and Runx. Both transcription factor families can be modulated by ERK and have been implicated in MMP-13 gene expression. TPA stimulated ERK-dependent increases in c-Fos protein and the c-Fos content of AP-1 complexes. MMP-13 promoter studies indicated that TPA requires AP-1, but not Runx, to induce MMP-13 gene expression. These studies show that in mouse keratinocytes MMP-13 gene expression can be induced through a Runx-independent pathway that involves the ERK-dependent modulation of AP-1.