Post-Acquisition Hyperpolarized 29Silicon Magnetic Resonance Image Processing for Visualization of Colorectal Lesions Using a User-Friendly Graphical Interface.

Medical imaging devices often use automated processing that creates and displays a self-normalized image. When improperly executed, normalization can misrepresent information or result in an inaccurate analysis. In the case of diagnostic imaging, a false positive in the absence of disease, or a negative finding when disease is present, can produce a detrimental experience for the patient and diminish their health prospects and prognosis. In many clinical settings, a medical technical specialist is trained to operate an imaging device without sufficient background information or understanding of the fundamental theory and processes involved in image creation and signal processing. Here, we describe a user-friendly image processing algorithm that mitigates user bias and allows for true signal to be distinguished from background. For proof-of-principle, we used antibody-targeted molecular imaging of colorectal cancer (CRC) in a mouse model, expressing human MUC1 at tumor sites. Lesion detection was performed using targeted magnetic resonance imaging (MRI) of hyperpolarized silicon particles. Resulting images containing high background and artifacts were then subjected to individualized image post-processing and comparative analysis. Post-acquisition image processing allowed for co-registration of the targeted silicon signal with the anatomical proton magnetic resonance (MR) image. This new methodology allows users to calibrate a set of images, acquired with MRI, and reliably locate CRC tumors in the lower gastrointestinal tract of living mice. The method is expected to be generally useful for distinguishing true signal from background for other cancer types, improving the reliability of diagnostic MRI.

SULF1 suppresses Wnt3A-driven growth of bone metastatic prostate cancer in perlecan-modified 3D cancer-stroma-macrophage triculture models.

Bone marrow stroma influences metastatic prostate cancer (PCa) progression, latency, and recurrence. At sites of PCa bone metastasis, cancer-associated fibroblasts and tumor-associated macrophages interact to establish a perlecan-rich desmoplastic stroma. As a heparan sulfate proteoglycan, perlecan (HSPG2) stores and stabilizes growth factors, including heparin-binding Wnt3A, a positive regulator of PCa cell growth. Because PCa cells alone do not induce CAF production of perlecan in the desmoplastic stroma, we sought to discover the sources of perlecan and its growth factor-releasing modifiers SULF1, SULF2, and heparanase in PCa cells and xenografts, bone marrow fibroblasts, and macrophages. SULF1, produced primarily by bone marrow fibroblasts, was the main glycosaminoglycanase present, a finding validated with primary tissue specimens of PCa metastases with desmoplastic bone stroma. Expression of both HSPG2 and SULF1 was concentrated in αSMA-rich stroma near PCa tumor nests, where infiltrating pro-tumor TAMs also were present. To decipher SULF1's role in the reactive bone stroma, we created a bone marrow biomimetic hydrogel incorporating perlecan, PCa cells, macrophages, and fibroblastic bone marrow stromal cells. Finding that M2-like macrophages increased levels of SULF1 and HSPG2 produced by fibroblasts, we examined SULF1 function in Wnt3A-mediated PCa tumoroid growth in tricultures. Comparing control or SULF1 knockout fibroblastic cells, we showed that SULF1 reduces Wnt3A-driven growth, cellularity, and cluster number of PCa cells in our 3D model. We conclude that SULF1 can suppress Wnt3A-driven growth signals in the desmoplastic stroma of PCa bone metastases, and SULF1 loss favors PCa progression, even in the presence of pro-tumorigenic TAMs.

Perlecan domain I gradients establish stable biomimetic heparin binding growth factor gradients for cell migration in hydrogels.

Growth factor gradients orchestrate many biological processes including organogenesis, wound healing, cancer invasion, and metastasis. Heparin-binding growth factor (HBGF) gradients are established in living systems by proteoglycans including the extracellular matrix heparan sulfate proteoglycan, perlecan/HSPG2. Three potential HBGF-binding glycosaminoglycan attachment sites occur in N-terminal domain I of perlecan's five domains. Our overarching goal was to form stable, biomimetic non-covalently bound HBGF gradients surrounding cells encapsulated in hyaluronate-based hydrogels by first establishing perlecan domain I (PlnD1) gradients. A versatile multichannel gradient maker device (MGMD) was designed and 3D printed, then used to create desired gradients of microparticles in hydrogels. Next, we used the device to covalently incorporate gradients of PEGylated PlnD1 in hydrogels with high-low-high or high-medium-low concentrations across the hydrogel width. Fluorescently-labeled fibroblast growth factor-2 was delivered to hydrogels in phosphate-buffered saline and allowed to electrostatically bind to the covalently pre-incorporated PlnD1, producing stable non-covalent HBGF gradients. To test cell viability after flow through the MGMD, delicate primary human salivary stem/progenitor cells were encapsulated in gradient hydrogels where they showed high viability and continued to grow. Next, to test migratory behavior in response to HBGF gradients, two cell types, preosteoblastic MC3T3-E1 cell line and breast cancer cell line MDA-MB-231 were encapsulated in or adjacent to PlnD1-modified hydrogels. Both cell lines migrated toward HBGFs bound to PlnD1. We conclude that establishing covalently-bound PlnD1 gradients in hydrogels provides a new means to establish physiologically-relevant gradients of HBGFs that are useful for a variety of applications in tissue engineering and cancer biology. STATEMENT OF SIGNIFICANCE: Gradients of heparin binding growth factors (HBGFs) direct cell behavior in living systems. HBGFs bind electrostatically to gradients of HS proteoglycans in the extracellular matrix creating HBGF gradients. We recreated HBGF gradients in physiological hyaluronate-based hydrogels using a 3D-printed multichannel gradient maker device (MGMD) that created gradients of HS proteoglycan-derived perlecan/HSPG2 domain I. We demonstrated the ability of a variety of cells, including primary salivary stem/progenitor cells, pre-osteoblastic cells and an invasive breast cancer cell line, to be co-encapsulated in gradient hydrogels by flowing them together through the MGMD. The versatile device and the ability to create HBGF gradients in hydrogels for a variety of applications is innovative and of broad utility in both cancer biology and tissue engineering applications.

PPARγ Modulation of Cytokine-Stimulated MUC16 (CA125) Expression in Breast and Ovarian Cancer-Derived Cells.

CA125 is serum tumor marker consisting of an epitope carried by a portion of the extremely large (>3 MDa), heavily glycosylated cell surface transmembrane mucin, MUC16. In malignancies, membrane bound mucins lose their polarized distribution, become aberrantly over-expressed and protect tumor cells from the actions of chemotherapeutic agents as well as the immune system. Previously, we described stimulation of MUC16 expression by the proinflammatory cytokines, tumor necrosis factor α (TNFα) and interferon γ (IFNγ), in breast and ovarian cancer cells and tissues. Herein, we show that PPARγ modulates cytokine-stimulated MUC16 in a complex manner: at low concentrations (<10 µM) rosiglitazone further potentiates cytokine-driven MUC16 expression while at high concentrations (>20 µM) rosiglitazone antagonizes cytokine stimulation. Rosiglitazone actions were fully reversible by the PPARγ antagonist, GW9662. Furthermore, siRNA-mediated PPARγ knockdown also prevented a large portion of high dose rosiglitazone suppression of MUC16 expression indicating that rosiglitazone inhibition is largely PPARγ-dependent. Cytokines greatly (>75%) suppressed PPARγ expression. Conversely, PPARγ activation by rosiglitazone at either low or high concentrations greatly (>75%) suppressed NFκB/p65 expression. NFκB/p65 expression was largely preserved in the presence of cytokines at low, but not high, rosiglitazone concentrations accounting for the different concentration dependent effects on MUC16 expression. Collectively, these studies demonstrate that PPARγ is an important modulator of MUC16 expression. The ability to deliver high doses of PPARγ agonists to MUC16-expressing tumors offers an avenue to reduce expression of this protective glycoprotein and increase tumor sensitivity to killing by chemotherapeutic drugs and the immune system. J. Cell. Biochem. 118: 163-171, 2017. © 2016 Wiley Periodicals, Inc.

MUC1 stimulates EGFR expression and function in endometrial cancer.

The current standard of care for endometrial cancer patients involves hysterectomy with adjuvant radiation and chemotherapy, with no effective treatment for advanced and metastatic disease. MUC1 is a large, heavily glycosylated transmembrane protein that lubricates and protects cell surfaces and increases cellular signaling through the epidermal growth factor receptor (EGFR). We show for the first time that MUC1 stimulates EGFR expression and function in endometrial cancer. siRNA knockdown and CRISPR/Cas knockout of MUC1 reduced EGFR gene expression, mRNA, protein levels and signaling. MUC1 bound strongly to two regions of the EGFR promoter: -627/-511 and -172/-64. MUC1 knockout also reduced EGFR-dependent proliferation in two dimensional culture, as well as growth and survival in three dimensional spheroid cultures. MUC1 knockout cells were more sensitive to the EGFR inhibitor, lapatinib. Finally, MUC1 and EGFR co-expression was associated with increased cellular proliferation in human endometrial tumors. These data demonstrate the importance of MUC1-driven EGFR expression and signaling and suggest dual-targeted therapies may provide improved response for endometrial tumors.

Tumor necrosis factor-α and interferon-γ stimulate MUC16 (CA125) expression in breast, endometrial and ovarian cancers through NFκB.

Transmembrane mucins (TMs) are restricted to the apical surface of normal epithelia. In cancer, TMs not only are over-expressed, but also lose polarized distribution. MUC16/CA125 is a high molecular weight TM carrying the CA125 epitope, a well-known molecular marker for human cancers. MUC16 mRNA and protein expression was mildly stimulated by low concentrations of TNFα (2.5 ng/ml) or IFNγ (20 IU/ml) when used alone; however, combined treatment with both cytokines resulted in a moderate (3-fold or less) to large (> 10-fold) stimulation of MUC16 mRNA and protein expression in a variety of cancer cell types indicating that this may be a general response. Human cancer tissue microarray analysis indicated that MUC16 expression directly correlates with TNFα and IFNγ staining intensities in certain cancers. We show that NFκB is an important mediator of cytokine stimulation of MUC16 since siRNA-mediated knockdown of NFκB/p65 greatly reduced cytokine responsiveness. Finally, we demonstrate that the 250 bp proximal promoter region of MUC16 contains an NFκB binding site that accounts for a large portion of the TNFα response. Developing methods to manipulate MUC16 expression could provide new approaches to treating cancers whose growth or metastasis is characterized by elevated levels of TMs, including MUC16.

Transmembrane Mucin Expression and Function in Embryo Implantation and Placentation.

Transmembrane mucins (TMs) are extremely large, complex glycoproteins that line the apical surfaces of simple epithelia including those of the female reproductive tract. TMs provide a physical barrier consistent with their role as part of the innate immune system. This barrier function must be overcome in the context of embryo implantation to permit blastocyst attachment. Three major TMs have been identified in uterine epithelia of multiple species: MUC1, MUC4, and MUC16. MUC1 has been found in all species studied to date, whereas expression of MUC4 and MUC16 have been less well studied and may be species specific. The strategies for removing mucins to permit embryo attachment also vary in a species-specific way and include both hormonal suppression of TM gene expression and membrane clearance via cell surface proteases. Studies emerging from the cancer literature indicate that TMs can modulate a surprisingly wide variety of signal transduction processes. Furthermore, various cell surface proteins have been identified that bind either the oligosaccharide or protein motifs of TMs suggesting that these molecules may support cell attachment in some contexts, including trophoblast interactions with cells of the immune system. The intimate association of TMs at sites of embryo-maternal interaction and the varied functions these complex molecules can play make them key players in embryo implantation and placentation processes.

Multilayered, Hyaluronic Acid-Based Hydrogel Formulations Suitable for Automated 3D High Throughput Drug Screening of Cancer-Stromal Cell Cocultures.

Validation of a high-throughput compatible 3D hyaluronic acid hydrogel coculture of cancer cells with stromal cells. The multilayered hyaluronic acid hydrogels improve drug screening predictability as evaluated with a panel of clinically relevant chemotherapeutics in both prostate and endometrial cancer cell lines compared to 2D culture.

Cytokine stimulation of MUC4 expression in human female reproductive tissue carcinoma cell lines and endometrial cancer.

MUC4, a transmembrane glycoprotein, interferes with cell adhesion, and promotes EGFR signaling in cancer. Studies in rat models have demonstrated steroid hormonal regulation of endometrial MUC4 expression. In this study, qRT-PCR screening of mouse tissues determined that Muc4 mRNA also was robustly expressed in mouse uteri. Previous studies from our labs have demonstrated MUC4 mRNA was expressed at levels <1% of MUC1 mRNA in human endometrium and endometriotic tissue. Multiple human endometrial adenocarcinoma cell lines were assayed for MUC4 mRNA expression revealing extremely low basal expression in the Ishikawa, RL-95-2, AN3CA, and KLE lines. Moderate to high expression was observed in HEC50 and HEC-1A cells. MUC4 mRNA expression was not affected by progesterone and/or estrogen treatment, but was greatly stimulated at both mRNA and protein levels by proinflammatory cytokines (IFN-γ and TNF-α), particularly when used in combination. In endometrial tissue, MUC4 mRNA levels did not change significantly between normal or cancerous samples; although, a subset of patients with grade 1 and 2 tumors displayed substantially higher expression. Likewise, immunostaining of human endometrial adenocarcinoma tissues revealed little to no staining in many patients (low MUC4), but strong staining in some patients (high MUC4) independent of cancer grade. In cases where staining was observed, it was heterogeneous with some cells displaying robust MUC4 expression and others displaying little or no staining. Collectively, these observations demonstrate that while MUC4 is highly expressed in the mouse uterus, it is not a major mucin in normal human endometrium. Rather, MUC4 is a potential marker of endometrial adenocarcinoma in a subset of patients.

MUC1 expression in fallopian tubes of women with hydrosalpinx.

OBJECTIVE: To analyze the expression of MUC1 in Fallopian tubes with or without hydrosalpinx, using four different types of antibody. STUDY DESIGN: In a case-control study, immunohistochemical expression of MUC1 was examined in Fallopian tubes derived from women with hydrosalpinx (n=10) and normal controls (n=10). Four different antibodies were used for the detection of both extracellular (214D4, HMFG1, VPM654) and intracellular (EPR1023) MUC1 epitopes. Staining intensity was measured with ImageJ software. Expression of MUC1 mRNA was quantified by quantitative RT-PCR. Statistical analysis was performed with Student t-test (mean ± SD) and Mann-Whitney U-test (median [range]). RESULTS: The mean (±SD) and median [range] intensity of MUC1 in controls vs. hydrosalpinx were: 214D4-67.5 ± 11.3 vs. 74.8 ± 14.69 (P=0.22); HMFG1-95.3 [642-1079] vs. 97.0 [502-1550] (P=0.91); VPM654-41.1 [314-914] vs. 46.0 [390-1424] (P=0.1); EPR1023-24.7 ± 7.3 vs. 57.4 ± 31.3 (P=0.01). MUC1 mRNA was 0.16 [008-05] vs. 0.09 [005-019] (P=0.06). Ectodomains and mRNA of MUC1 are unchanged in tubes from hydrosalpinx patients. In contrast, immunodetection of the MUC1 cytoplasmic tail is enhanced in tubes from hydrosalpinx. CONCLUSION: Fallopian tubes with hydrosalpinx have a selective accumulation of MUC1 cytoplasmic tail, but not difference in the ectodomain.

Transcriptional activation by NFκB increases perlecan/HSPG2 expression in the desmoplastic prostate tumor microenvironment.

Perlecan/HSPG2, a heparan sulfate proteoglycan typically found at tissue borders including those separating epithelia and connective tissue, increases near sites of invasion of primary prostatic tumors as previously shown for other proteins involved in desmoplastic tissue reaction. Studies of prostate cancer cells and stromal cells from both prostate and bone, the major site for prostate cancer metastasis, showed that cancer cells and a subset of stromal cells increased production of perlecan in response to cytokines present in the tumor microenvironment. In silico analysis of the HSPG2 promoter revealed two conserved NFκB binding sites, in addition to the previously reported SMAD3 binding sites. By systematically transfecting cells with a variety of reporter constructs including sequences up to 2.6 kb from the start site of transcription, we identified an active cis element in the distal region of the HSPG2 promoter, and showed that it functions in regulating transcription of HSPG2. Treatment with TNF-α and/or TGFß1 identified TNF-α as a major cytokine regulator of perlecan production. TNF-α treatment also triggered p65 nuclear translocation and binding to the HSPG2 regulatory region in stromal cells and cancer cells. In addition to stromal induction of perlecan production in the prostate, we identified a matrix-secreting bone marrow stromal cell type that may represent the source for increases in perlecan in the metastatic bone marrow environment. These studies implicate perlecan in cytokine-mediated, innate tissue responses to cancer cell invasion, a process we suggest reflects a modified wound healing tissue response co-opted by prostate cancer cells.

Border patrol: insights into the unique role of perlecan/heparan sulfate proteoglycan 2 at cell and tissue borders.

The extracellular matrix proteoglycan (ECM) perlecan, also known as heparan sulfate proteoglycan 2 or HSPG2, is one of the largest (>200 nm) and oldest (>550 M years) extracellular matrix molecules. In vertebrates, perlecan's five-domain structure contains numerous independently folding modules with sequence similarities to other ECM proteins, all connected like cars into one long, diverse complex train following a unique N-terminal domain I decorated with three long glycosaminoglycan chains, and an additional glycosaminoglycan attachment site in the C-terminal domain V. In lower invertebrates, perlecan is not typically a proteoglycan, possessing the majority of the core protein modules, but lacking domain I where the attachment sites for glycosaminoglycan chains are located. This suggests that uniting the heparan sulfate binding growth factor functions of domain I and the core protein functions of the rest of the molecule in domains II-V occurred later in evolution for a new functional purpose. In this review, we surveyed several decades of pertinent literature to ask a fundamental question: Why did nature design this protein uniquely as an extraordinarily long multifunctional proteoglycan with a single promoter regulating expression, rather than separating these functions into individual proteins that could be independently regulated? We arrived at the conclusion that the concentration of perlecan at functional borders separating tissues and tissue layers is an ancient key function of the core protein. The addition of the heparan sulfate chains in domain I likely occurred as an additional means of binding the core protein to other ECM proteins in territorial matrices and basement membranes, and as a means to reserve growth factors in an on-site depot to assist with rapid repair of those borders when compromised, such as would occur during wounding. We propose a function for perlecan that extends its role from that of an extracellular scaffold, as we previously suggested, to that of a critical agent for establishing and patrolling tissue borders in complex tissues in metazoans. We also propose that understanding these unique functions of the individual portions of the perlecan molecule can provide new insights and tools for engineering of complex multi-layered tissues including providing the necessary cues for establishing neotissue borders.

Activated EGFR stimulates MUC1 expression in human uterine and pancreatic cancer cell lines.

MUC1 is a large cell surface mucin glycoprotein that plays diverse roles in both normal and tumor cell biology. These roles include mucosal hydration and protection, inhibition of embryo implantation, protection of tumor cells from the immune system and reduction of cytotoxic drug uptake. Similarly, the EGFR family of cell surface receptors drives many normal developmental processes as well as various aspects of tumor growth and gene expression. EGFR family members have been demonstrated to form complexes with MUC1 in various cellular contexts. Nonetheless, the role that EGFR activation plays in modulating MUC1 levels has not been considered. In this study, we demonstrate that activated EGFR drives high level MUC1 expression in multiple cell lines of uterine adenocarcinoma and pancreatic cancer origins. In some cells, addition of exogenous EGFR ligands (EGF or HB-EGF) elevates MUC1 levels while addition of the EGFR tyrosine kinase inhibitor, AG1478, reduces MUC1 levels. The thiazolidinedione, rosiglitazone, previously shown to reduce progesterone-stimulated MUC1 expression, also blocks EGFR ligand-driven MUC1 expression. This activity was observed at relatively high rosiglitazone concentrations (above 10 µM) and appeared to be largely PPARγ independent indicating a novel utility of this drug to reduce mucin-expression in various tumor settings. Collectively, these data demonstrate that: (1) activation of EGFR stimulates MUC1 expression in multiple cellular contexts and (2) it may be possible to develop useful interventions to reduce MUC1 expression as a complementary strategy for tumor therapy.

The MUC1 Ectodomain: A Novel and Efficient Target for Gold Nanoparticle Clustering and Vapor Nanobubble Generation.

MUC1 is a large, heavily glycosylated transmembrane glycoprotein that is proposed to create a protective microenvironment in many adenocarcinomas. Here we compare MUC1 and the well studied cell surface receptor target, EGFR, as gold nanoparticle (AuNP) targets and their subsequent vapor nanobubble generation efficacy in the human epithelial cell line, HES. Although EGFR and MUC1 were both highly expressed in these cells, TEM and confocal images revealed MUC1 as a superior target for nanoparticle intracellular accumulation and clustering. The MUC1-targeted AuNP intracellular clusters also generated significantly larger vapor nanobubbles. Our results demonstrate the promising opportunities MUC1 offers to improve the efficacy of targeted nanoparticle based approaches.

Improved cellular specificity of plasmonic nanobubbles versus nanoparticles in heterogeneous cell systems.

The limited specificity of nanoparticle (NP) uptake by target cells associated with a disease is one of the principal challenges of nanomedicine. Using the threshold mechanism of plasmonic nanobubble (PNB) generation and enhanced accumulation and clustering of gold nanoparticles in target cells, we increased the specificity of PNB generation and detection in target versus non-target cells by more than one order of magnitude compared to the specificity of NP uptake by the same cells. This improved cellular specificity of PNBs was demonstrated in six different cell models representing diverse molecular targets such as epidermal growth factor receptor, CD3 receptor, prostate specific membrane antigen and mucin molecule MUC1. Thus PNBs may be a universal method and nano-agent that overcome the problem of non-specific uptake of NPs by non-target cells and improve the specificity of NP-based diagnostics, therapeutics and theranostics at the cell level.

Transmembrane mucins as novel therapeutic targets.

Membrane-tethered mucin glycoproteins are abundantly expressed at the apical surfaces of simple epithelia, where they play important roles in lubricating and protecting tissues from pathogens and enzymatic attack. Notable examples of these mucins are MUC1, MUC4 and MUC16 (also known as cancer antigen 125). In adenocarcinomas, apical mucin restriction is lost and overall expression is often highly increased. High-level mucin expression protects tumors from killing by the host immune system, as well as by chemotherapeutic agents, and affords protection from apoptosis. Mucin expression can increase as the result of gene duplication and/or in response to hormones, cytokines and growth factors prevalent in the tumor milieu. Rises in the normally low levels of mucin fragments in serum have been used as markers of disease, such as tumor burden, for many years. Currently, several approaches are being examined that target mucins for immunization or nanomedicine using mucin-specific antibodies.

Paracrine factors produced by bone marrow stromal cells induce apoptosis and neuroendocrine differentiation in prostate cancer cells.

BACKGROUND: Preferential bony metastasis of human prostate cancer (PCa) cells contributes to disease mortality and morbidity. Local factors in bone stromal extracellular matrix microenvironment affect tumor growth through paracrine interactions between tumor and stromal cells. METHODS: Using co-culture and medium transfer, we used several methods to assess interactions between PCa and bone stromal cells using three PCa cell lines: PC3, LNCaP, and the LNCaP derivative, C4-2B. RESULTS: Co-culture of LNCaP and C4-2B cells with bone marrow stromal cell lines, HS27a and HS5, decreased cell number, as did culture with conditioned medium (CM) harvested from these two cell lines suggesting a soluble paracrine factor was responsible. PC3 cell growth was unaffected. CM harvested from bone stromal cell lines triggered apoptosis in LNCaP and C4-2B cell lines, but not in PC3 cells. Surviving C4-2B cells grown in bone stromal cell CM over several days were growth arrested, suggesting presence of a growth inhibitor. Apoptosis induced by CM was dose-dependent. Flow cytometry demonstrated that over a 5-day culture period in stromal cell CM, LNCaP, and C4-2B cell lines, but not PC3 cells, underwent greater apoptosis than parallel cultures in SF medium. The LNCaP and C4-2B cells showed morphology and biomarker expression consistent with transdifferentiation towards a neuroendocrine phenotype after exposure to stromal cell CM. CONCLUSIONS: The reactive bone stromal microenvironment initially is hostile to PCa cells producing widespread apoptosis. Activation of transdifferentiation in a subset of apoptotic resistant cells may support phenotypic adaptation during disease progression in bone, eventually favoring lethal disease.

Cytokine and progesterone receptor interplay in the regulation of MUC1 gene expression.

Mucin 1 (MUC1), a transmembrane mucin expressed at the apical surface of uterine epithelia, is a barrier to microbial infection and enzymatic attack. MUC1 loss at implantation sites appears to be required to permit embryo attachment and implantation in most species. MUC1 expression is regulated by progesterone (P) and proinflammatory cytokines, including TNFα and interferon γ (IFNγ). TNFα and IFNγ are highly expressed in uterine tissues under conditions where MUC1 expression is also high and activate MUC1 expression via their downstream transcription factors, nuclear factor (NF) κB and signal transducers and activators of transcription. P receptor (PR) regulates MUC1 gene expression in a PR isoform-specific fashion. Here we demonstrate that interactions among PR isoforms and cytokine-activated transcription factors cooperatively regulate MUC1 expression in a human uterine epithelial cell line, HES. Low doses of IFNγ and TNFα synergistically stimulate MUC1 promoter activity, enhance PRB stimulation of MUC1 promoter activity and cooperate with PRA to stimulate MUC1 promoter activity. Cooperative stimulation of MUC1 promoter activity requires the DNA-binding domain of the PR isoforms. MUC1 mRNA and protein expression is increased by cytokine and P treatment in HES cells stably expressing PRB. Using chromatin immunoprecipitation assays, we demonstrate efficient recruitment of NFκB, p300, SRC3 (steroid receptor coactivator 3), and PR to the MUC1 promoter. Collectively, our studies indicate a dynamic interplay among cytokine-activated transcription factors, PR isoforms and transcriptional coregulators in modulating MUC1 expression. This interplay may have important consequences in both normal and pathological contexts, e.g. implantation failure and recurrent miscarriages.

Peroxisome proliferator-activated receptor gamma activation inhibits progesterone-stimulated human MUC1 expression.

Mucin 1 (MUC1) is a type I transmembrane glycoprotein abundantly expressed on nearly all epithelial tissues and overexpressed by many cancer cells. Previous studies from our lab showed that progesterone receptor (PR)B is a strong stimulator of MUC1 gene expression. It is reported that liganded peroxisome proliferator-activated receptor gamma (PPARgamma) stimulates Muc1 expression in murine trophoblast. Here, we demonstrate that although the PPARgamma ligand, rosiglitazone, stimulates the murine Muc1 promoter in HEC1A, a human uterine epithelial cell line, rosiglitazone alone, has no significant effect on basal human MUC1 promoter activity. In fact, rosiglitazone treatment antagonizes progesterone-stimulated human MUC1 promoter activity and protein expression in two human uterine epithelial cell lines and T47D human breast cancer cells. This response is antagonized by the PPARgamma antagonist, GW9662, as well as a dominant-negative form of PPARgamma, demonstrating the response is mediated by PPARgamma. Additional studies indicate that PPARgamma activation does not change PR binding to the MUC1 promoter but generally antagonizes progesterone activity by stimulating PRB degradation and inhibiting progesterone-induced PRB phosphorylation. Collectively, these studies indicate that PPARgamma activation inhibits PRB activity through both acute (phosphorylation) and long-term (PRB degradation) pathways.

Expression of human MUC1 during early pregnancy in the human MUC1 transgenic mouse model.

Embryo implantation involves direct interaction of the blastocyst with the luminal epithelium of the receptive uterus. MUC1, a transmembrane mucin expressed at the apical surface of uterine epithelia, acts as a barrier to microbial infection and enzymatic attack. Loss of MUC1 is believed to be a prerequisite for a functionally receptive uterus across many species. Human and murine MUC1 regulation by steroid hormones displays important differences. Estrogen (E2) stimulates MUC1 expression in mice, and progesterone (P4) antagonizes E2 action in this regard. MUC1 expression is severely reduced during the receptive uterine state in mice. In contrast, human MUC1 expression is maximal at the receptive or midluteal phase, when P4 levels are high. No information is available regarding regulation of human MUC1 in vivo at the site of embryo attachment. Our aim was to better understand regulation of human MUC1 during early pregnancy in vivo. For this purpose, we used a transgenic mouse carrying full-length human MUC1 gene (Tg(MUC1)79.24Gend) as well as endogenous MUC1 as a model system. Human MUC1 was detected by real-time RT-PCR, Western blotting, and immunohistochemistry during early pregnancy. Our data indicate that human MUC1 persists at reduced (20% relative to Day 1 postcoitum) levels in receptive-phase uteri, including the site of embryo attachment. In contrast, mouse MUC1 was much more severely (>98% relative to Day 1 postcoitum) reduced in the same context. These observations are consistent with distinct regulation between the human and mouse genes. Because these genes are expressed in the same transcriptional context (i.e., mouse uterine epithelia), structural differences between human and murine genes must account for these differences in MUC1 regulation.