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Background The Zc3h8 gene encodes a protein with three zinc finger motifs in the C-terminal region. The protein has been identified as a component of the Little Elongation Complex, involved in transcription of small nuclear RNAs. ZC3H8 is overexpressed in a number of human and mouse breast cancer cell lines, and elevated mRNA levels are associated with a poorer prognosis for women with breast cancer. Methods We used RNA silencing to decrease levels of expression in mouse mammary tumor cells and overexpression of ZC3H8 in cells derived from the normal mouse mammary gland. We measured characteristics of cell behavior in vitro, including proliferation, migration, invasion, growth in soft agar, and spheroid growth. We assessed the ability of these cells to form tumors in syngeneic BALB/c mice. ZC3H8 protein was visualized in cells using confocal microscopy. Results Tumor cells with lower ZC3H8 expression exhibited decreased proliferation rates, slower migration, reduced ability to invade through a basement membrane, and decreased anchorage independent growth in vitro. Cells with lower ZC3H8 levels formed fewer and smaller tumors in animals. Overexpression of ZC3H8 in non-tumorigenic COMMA-D cells led to an opposite effect. ZC3H8 protein localized to both PML bodies and Cajal bodies within the nucleus. ZC3H8 has a casein kinase 2 (CK2) phosphorylation site near the N-terminus, and a CK2 inhibitor caused the numerous PML bodies and ZC3H8 to coalesce to a few larger bodies. Removal of the inhibitor restored PML bodies to their original state. A mutant ZC3H8 lacking the predicted CK2 phosphorylation site showed localization and numbers of ZC3H8/PML bodies similar to wild type. In contrast, a mutant constructed with a glutamic acid in place of the phosphorylatable threonine showed dramatically increased numbers of smaller nuclear foci. Conclusions These experiments demonstrate that Zc3h8 expression contributes to aggressive tumor cell behavior in vitro and in vivo. Our studies show that ZC3H8 integrity is key to maintenance of PML bodies. The work provides a link between the Little Elongation Complex, PML bodies, and the cancer cell phenotype. Electronic supplementary material The online version of this article (10.1186/s12885-018-4674-1) contains supplementary material, which is available to authorized users.

Zc3h8 encodes a zinc finger protein that is amplified in 2-6% of human breast tumors. Higher expression of Zc3h8 also results in lower rates of disease-free survival. Amplification of Zc3h8 expression has been shown to increase cell proliferation, migration, invasion and tumor growth in mice. Conversely, decreased expression alters cell behavior by decreasing cell proliferation, migration, invasion, and tumor growth in mice. Zc3h8 comprises three CCCH non-classical zinc finger domains at the C-terminus of the protein that we hypothesize bind to RNA. The interactions between Zc3h8 and nucleic acids may be important for the localization and/or function of Zc3h8 and therefore may be important for the cellular phenotypes observed when the gene is over or under expressed in cells. We used in vitro and in vivo assays to demonstrate that mutations in any of the three zinc finger domains results in a protein that fails to rescue aggressive cellular phenotypes in cells with reduced expression of Zc3h8, while the wild type protein can rescue these phenotypes. This implies that all three zinc finger domains are required for Zc3h8 function in promoting rapid and aggressive cell proliferation. In addition to zinc finger domains, Zc3h8 also contains a casein kinase-2 (CK2) phosphorylation site that may be important for protein localization and/or activation. We have previously shown that CK2 inhibitors alter Zc3h8 localization to nuclear PML Bodies. Mouse cells overexpressing Zc3h8 with a Thr to Ala (T32A) change that prevents CK2 phosphorylation grow very slowly in culture and cannot grow in more challenging 3D or soft agar growth chambers. However, Zc3h8 with a Thr to Glu (T32E) change that mimics constitutive CK2 phosphorylation results in rapidly growing cells that surpass overexpression of Zc3h8 wild type. Zc3h8 phospho-mutants also have altered localization with Zc3h8 T32E becoming diffuse in the nucleus and T32A localizing to nuclear foci. These data suggest that the phosphorylation state of Zc3h8 and its ability to bind nucleic acids via zinc finger domains are critical for the oncogenic behavior that results from amplified expression. We also hypothesize that Zc3h8 has an important role in establishing or maintaining PML Bodies involved in genome maintenance, transcription, and/or post-transcription regulation. Citation Format: John A. Schmidt, Tyler Doan, Emanuel Irizarry, Emily Harris, Keith G. Danielson, Janice E. Knepper. Molecular dissection of Zc3h8 functional domains and the implications for the oncogenic phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1713.

The zinc finger protein Zc3h8 is overexpressed in many mouse and human solid tumors. We have shown that increased expression of Zc3h8 in cultured mouse mammary cells is associated with increased proliferation rate, increased motility and invasiveness, increased capacity for growth in three dimensions, and increased capacity for tumor formation. Partial knockdown of Zc3h8 expression from mouse mammary tumor cells has the opposite effect. The Zc3h8 protein localizes to both PML bodies and Cajal bodies in the nucleus and features of this localization are altered by treatment of cells with a casein kinase 2 inhibitor, TBB. We have embarked upon an analysis of the contributions of structural features of the Zc3h8 protein through mutagenesis of the gene at key positions. The casein kinase 2 phosphorylation site at position T32 was mutated to either an alanine to prevent phosphorylation, or to glutamic acid to mimic constitutive phosphorylation. Each of the three zinc fingers in the C-terminal region was mutated to replace one cysteine residue. The mutations were introduced into a variant of the zc3h8 gene that has 6 mutations introduced at synonymous positions in order to escape knockdown by RNAi, although the protein sequence generated is identical to the original Zc3h8. Each of the mutant constructs was introduced into mouse mammary tumor cells with knockdown of the endogenous Zc3h8. Mutation of the phosphorylation site T32A led to the formation of fewer, although larger PML bodies, with continued ZC3h8 co-localization. The T32E mutant, with constitutive negative charge at this position led to the formation of a large number of small nuclear foci. Mutant constructs were stably transfected into tumor cells with knockdown of endogenous Zc3h8, and behavior of the mutants was compared to the behavior of cells transfected with the synonymously mutated gene construct. Disruption of each of the zinc fingers individually led to decreased proliferation rate, decreased growth as spheroids or in soft agar, and decreased invasion through Matrigel relative to the synonymous Zc3h8. Tumor cells with Zc3h8 knockdown failed to form tumors in syngeneic BALB/c mice within 10 weeks, while cells transfected with a negative control vector formed tumors in all animals. Rescue of Zc3h8 expression with the synonymous mutant led to tumor formation in 50% of the animals, while no tumors formed from cells transfected with zinc finger mutants. We conclude that the zinc fingers contribute to aggressive tumor cell behavior while the T32 phosphorylation site is essential for proper localization to nuclear bodies. Citation Format: John A. Schmidt, Emily R. Duffner, Emily M. Harris, Tyler Doan, Emanuel Irizarry, Keith G. Danielson, Janice E. Knepper. Structural analysis of features contributing to the oncogenic phenotype conferred by the zinc finger nuclear body protein Zc3h8 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2484.

Although a recent study has shown that skeletal tissues express ASICs, their function is unknown. We show that intervertebral disc cells express ASIC3; moreover, expression is uniquely regulated and needed for survival in a low pH and hypoeromsotic medium. These findings suggest that ASIC3 may adapt disc cells to their hydrodynamically stressed microenvironment. Introduction: The nucleus pulposus is an avascular, hydrated tissue that permits the intervertebral disc to resist compressive loads to the spine. Because the tissue is hyperosmotic and avascular, the pH of the nucleus pulposus is low. To determine the mechanisms by which the disc cells accommodate to the low pH and hypertonicity, the expression and regulation of the acid sensing ion channel (ASIC)3 was examined. Materials and Methods: Expression of ASICs in cells of the intervertebral disc was analyzed. To study its regulation, we cloned the 2.8-kb rat ASIC3 promoter and performed luciferase reporter assays. The effect of pharmacological inhibition of ASICs on disc cell survival was studied by measuring MTT and caspase-3 activities. Results: ASIC3 was expressed in discal tissues and cultured disc cells in vitro. Because studies of neuronal cells have shown that ASIC3 expression and promoter activity is induced by nerve growth factor (NGF), we examined the effect of NGF on nucleus pulposus cells. Surprisingly, ASIC3 promoter activity did not increase after NGF treatment. The absence of induction was linked to nonexpression of tropomyosin-related kinase A (TrkA), a high-affinity NGF receptor, although a modest expression of p75NTR was seen. When treated with p75NTR antibody or transfected with dominant negative-p75NTR plasmid, there was significant suppression of ASIC3 basal promoter activity. To further explore the downstream mechanism of control of ASIC3 basal promoter activity, we blocked p75NTR and measured phospho extracellular matrix regulated kinase (pERK) levels. We found that DN-p75NTR suppressed NGF mediated transient ERK activation. Moreover, inhibition of ERK activity by dominant negative-mitogen activated protein kinase kinase (DN-MEK) resulted in a dose-dependent suppression of ASIC3 basal promoter activity, whereas overexpression of constitutively active MEK1 caused an increase in ASIC3 promoter activity. Finally, to gain insight in the functional importance of ASIC3, we suppressed ASIC activity in nucleus pulposus cells. Noteworthy, under both hyperosmotic and acidic conditions, ASIC3 served to promote cell survival and lower the activity of the pro-apoptosis protein, caspase-3. Conclusions: Results of this study indicate that NGF serves to maintain the basal expression of ASIC3 through p75NTR and ERK signaling in discal cells. We suggest that ASIC3 is needed for adaptation of the nucleus pulposus and annulus fibrosus cells to the acidic and hyperosmotic microenvironment of the intervertebral disc.

The nucleus pulposus is an aggrecan-rich hydrated tissue that permits the intervertebral disc to resist compressive loads. Adaptation to loading is achieved through an elevation in disc osmolarity mediated by the numerous charged glycosoaminoglycan side chains of the aggrecan molecule. The goal of this investigation was to determine the functional role of the osmo-regulatory protein, TonEBP, in cells of the nucleus pulposus. We found that TonEBP and its downstream target genes were robustly expressed in the tissues of the disc. Above 330 mosmol/kg, cultured nucleus pulposus cells up-regulated target genes TauT, BGT-1, and SMIT; above 450 mosmol/kg, there was raised expression of HSP-70. In hypertonic media there was activation of TauT and heat shock protein-70 (HSP-70) reporter activity and increased binding of TonEBP to the TonE motif. When cells were transfected with the dominant-negative form of TonEBP (DN-TonEBP) there was suppression of TauT and HSP-70 reporter gene expression; pTonEBP enhanced reporter gene expression. Moreover, in hypertonic media, forced expression of DN-TonEBP induced apoptosis. We suppressed TonEBP using small interfering RNA technique and noted a decrease in TauT reporter activity in isotonic as well as hyperosmolar media. Finally, we report that the aggrecan promoter contains two conserved TonE motifs. To evaluate the importance of these motifs, we overexpressed DN-TonEBP and partially silenced TonEBP using small interfering RNA. Both approaches resulted in suppression of aggrecan promoter activity. It is concluded that TonEBP permits the disc cells to adapt to the hyperosmotic milieu while autoregulating the expression of molecules that generate the unique extracellular environment.

The utilization of adult stem cells in tissue engineering is a promising solution to the problem of tissue or organ shortage. Adult bone marrow derived mesenchymal stem cells (MSCs) are undifferentiated, multipotential cells which are capable of giving rise to chondrocytes when maintained in a three-dimensional culture and treated with members of the transforming growth factor-beta (TGF-beta) family of growth factors. In this study, we fabricated a nanofibrous scaffold (NFS) made of a synthetic biodegradable polymer, poly(-caprolactone) (PCL), and examined its ability to support in vitro chondrogenesis of MSCs. The electrospun PCL porous scaffold was constructed of uniform, randomly oriented nanofibers with a diameter of 700 nm, and structural integrity of this scaffold was maintained over a 21-day culture period. MSCs cultured in NFSs in the presence of TGF-beta1 differentiated to a chondrocytic phenotype, as evidenced by chondrocyte-specific gene expression and synthesis of cartilage-associated extracellular matrix (ECM) proteins. The level of chondrogenesis observed in MSCs seeded within NFSs was comparable to that observed for MSCs maintained as cell aggregates or pellets, a widely used culture protocol for studying chondrogenesis of MSCs in vitro. Due to the physical nature and improved mechanical properties of NFSs, particularly in comparison to cell pellets, the findings reported here suggest that the PCL NFS is a practical carrier for MSC transplantation, and represents a candidate scaffold for cell-based tissue engineering approaches to cartilage repair.

The in vitro culture of human trabecular bone-derived cells has served as a useful system for the investigation of the biology of osteoblasts. The recent discovery in our laboratory of the multilineage mesenchymal differentiation potential of cells derived from collagenase-treated human trabecular bone fragments has prompted further interest in view of the potential application of mesenchymal progenitor cells (MPCs) in the repair and regeneration of tissue damaged by disease or trauma. Similar to human MPCs derived from bone marrow, a clearer understanding of the variability associated with obtaining these bone-derived cells is required in order to optimize the design and execution of applicable studies. In this study, we have identified the presence of a CD73(+), STRO-1(+), CD105(+), CD34(-), CD45(-), CD144(-) cell population resident within collagenase-treated, culture-processed bone fragments, which upon migration established a homogeneous population of MPCs. Additionally, we have introduced a system of culturing these MPCs that best supports and maintains their optimal differentiation potential during long-term culture expansion. When cultured as described, the trabecular bone-derived cells display stem cell-like capabilities, characterized by a stable undifferentiated phenotype as well as the ability to proliferate extensively while retaining the potential to differentiate along the osteoblastic, adipocytic, and chondrocytic lineages, even when maintained in long-term in vitro culture.

The multilineage differentiation potential of adult tissue-derived mesenchymal progenitor cells (MPCs), such as those from bone marrow and trabecular bone, makes them a useful model to investigate mechanisms regulating tissue development and regeneration, such as cartilage. Treatment with transforming growth factor-beta (TGF-beta) superfamily members is a key requirement for the in vitro chondrogenic differentiation of MPCs. Intracellular signaling cascades, particularly those involving the mitogen-activated protein (MAP) kinases, p38, ERK-1, and JNK, have been shown to be activated by TGF-betas in promoting cartilage-specific gene expression. MPC chondrogenesis in vitro also requires high cell seeding density, reminiscent of the cellular condensation requirements for embryonic mesenchymal chondrogenesis, suggesting common chondro-regulatory mechanisms. Prompted by recent findings of the crucial role of the cell adhesion protein, N-cadherin, and Wnt signaling in condensation and chondrogenesis, we have examined here their involvement, as well as MAP kinase signaling, in TGF-beta1-induced chondrogenesis of trabecular bone-derived MPCs. Our results showed that TGF-beta1 treatment initiates and maintains chondrogenesis of MPCs through the differential chondro-stimulatory activities of p38, ERK-1, and to a lesser extent, JNK. This regulation of MPC chondrogenic differentiation by the MAP kinases involves the modulation of N-cadherin expression levels, thereby likely controlling condensation-like cell-cell interaction and progression to chondrogenic differentiation, by the sequential up-regulation and progressive down-regulation of N-cadherin. TGF-beta1-mediated MAP kinase activation also controls WNT-7A gene expression and Wnt-mediated signaling through the intracellular beta-catenin-TCF pathway, which likely regulates N-cadherin expression and subsequent N-cadherin-mediated cell-adhesion complexes during the early steps of MPC chondrogenesis.

The ZC3H8 gene encodes a protein with three zinc finger motifs in the C-terminal region, suggesting a role as an RNA binding protein, but whose specific function remains unclear. ZC3H8 protein localizes to both PML bodies and Cajal bodies within the nucleus, as confocal microscopy demonstrates colocalization of ZC3H8 with both PML and coilin proteins. ZC3H8 has a casein kinase 2 phosphorylation site in the N-terminal region, and treatment with a casein kinase 2 inhibitor causes the numerous PML bodies and their associated ZC3H8 to coalesce to a few larger bodies. Removal of the inhibitor restores PML bodies to their original state. We constructed a mutant of ZC3H8 that removed the predicted CK2 phosphorylation site by replacing the serine with an alanine residue. Cells transfected with this mutant had reduced numbers of PML bodies, similar to cells treated with the CK2 inhibitor. In contrast, a mutant constructed with a glutamic acid in place of the phosphorylatable serine was predicted to behave as a constitutive mutant. This mutant did not exhibit differences from wild type localization. These experiments suggest that ZC3H8 integrity is key to maintenance of PML bodies. Other studies have identified ZC3H8 as a component of the LEC (little elongation complex), and we found that another identified component, ICE2 (Narg) also localizes to PML bodies. The ZC3H8 gene is overexpressed in a number of human and mouse breast cancer cell lines, and elevated mRNA levels are associated with a poorer prognosis for women with breast cancer. We have used RNA silencing to decrease levels of expression in two independent mouse mammary tumor cell lines. Cells with lower ZC3H8 expression have dramatic phenotypic changes, showing decreased rates of proliferation, slower migration in wound healing assays, formation of fewer and smaller colonies in soft agar assays, and decreased ability to invade through a basement membrane in transwell assays. Further, ZC3H8-depleted cells have decreased rates of growth as spheroids and decreased growth in vivo in syngeneic mice. We developed an inducible vector driving expression of ZC3H8. Upon treatment with the inducer, doxycycline, cells assume a more aggressive phenotype. We introduced a synonymous mutant of ZC3H8 unable to be silenced by the RNAi into the knockdown cells, and rescued the aggressive phenotype of the original tumor cells. Overexpression of ZC3H8 in non-tumorigenic COMMA-D cells leads to a less pronounced, but opposite, increase in proliferation, motility, and invasion. We have engineered the synonymous ZC3H8 with disrupted zinc finger motifs, and preliminary results suggest that disruption of the first zinc finger is not sufficient to cause loss of the aggressive phenotype. We suggest that ZC3H8 is an oncogene with substantial effects on the cancer cell phenotype. These effects are hypothesized to be associated with ZC3H8 function in PML bodies. Citation Format: John A. Schmidt, Emily Duffner, Gerard Walker, Keith G. Danielson, Janice E. Knepper. ZC3H8 associates with PML bodies and influences aggressive tumor cell behavior [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2555. doi:10.1158/1538-7445.AM2017-2555

Perlecan, a modular heparan sulfate proteoglycan of basement membranes and cell surfaces, plays a crucial role in regulating the assembly of extracellular matrices and the binding of nutrients and growth factors to target cells. To achieve a molecular understanding of perlecan gene regulation, we isolated the 5′-flanking region and investigated its functional promoter activity and its response to cytokines. Transient cell transfection assays, using plasmid constructs harboring the perlecan promoter linked to the chloramphenicol acetyltransferase reporter gene, demonstrated that the largest ∼2.5-kilobase construct contained maximal promoter activity. This promoter region was functionally active in a variety of cells of diverse histogenetic origin, thus corroborating the widespread expression of this gene product. Stepwise 5′ deletion analyses demonstrated that the −461-base pair (bp) proximal promoter retained ∼90% of the total activity, and internal deletions confirmed that the most proximal sequence was essential for proper promoter activity. Nanomolar amounts of transforming growth factor-β induced 2-3-fold perlecan mRNA and protein core levels in normal human skin fibroblasts, and this induction was transcriptionally regulated; in contrast, tumor necrosis factor-α had no effect and was incapable of counteracting the effects of TGF-β. Using additional 5′ deletions and DNase footprinting analyses, we mapped the TGF-β responsive region to a sequence of 177 bp contained between −461 and −285. This region harbored a 14-bp element similar to a TGF-β-responsive element present in the promoters of collagen α1(I), α2(I), elastin, and growth hormone. Electrophoretic mobility shift assays and mutational analyses demonstrated that the perlecan TGF-β-responsive element bound specifically to TGF-β-inducible nuclear proteins with high affinity for NF-1 member(s) of transcription factors.

Decorin is a member of the expanding group of widely distributed small leucine-rich proteoglycans that are expected to play important functions in tissue assembly. We report that mice harboring a targeted disruption of the decorin gene are viable but have fragile skin with markedly reduced tensile strength. Ultrastructural analysis revealed abnormal collagen morphology in skin and tendon, with coarser and irregular fiber outlines. Quantitative scanning transmission EM of individual collagen fibrils showed abrupt increases and decreases in mass along their axes, thereby accounting for the irregular outlines and size variability observed in cross-sections. The data indicate uncontrolled lateral fusion of collagen fibrils in the decorindeficient mice and provide an explanation for the reduced tensile strength of the skin. These findings demonstrate a fundamental role for decorin in regulating collagen fiber formation in vivo.

The Zinc Finger CCCH-Type Containing 8 (zc3h8 or Fliz1) gene is overexpressed in many mouse and human breast cancer cell lines that we examined, although the exact function of this gene and the ZC3H8 protein are largely unknown. To characterize ZC3H8 protein, we localized it to distinct sub-nuclear domains that also contain markers for promyelocytic leukemia (PML) nuclear bodies and Cajal bodies. Furthermore, when cells are treated with a casein kinase-2 (CK2) inhibitor, ZC3H8 mislocalizes out of the nuclear bodies and diffuses into the nucleoplasm. CK2 is a known activator of transcription and a potential drug target. ZC3H8 contains a putative CK2 phosphorylation site at position T32, and may be regulated by this kinase. Based on this localization and other preliminary data, we hypothesize that ZC3H8 has a role in transcriptional regulation or RNA modification at these nuclear bodies. This hypothesis is supported by published studies that have found a role for ZC3H8 in the Little Elongation Complex (LEC), which transcribes snRNA involved in RNA splicing in nuclear bodies. Since many tumors and cancer cell lines overexpress zc3h8, we used stably expressed shRNA to knock down expression in two independent mouse breast cancer cell lines for in vitro and in vivo studies. Cells with reduced zc3h8 expression have decreased rates of proliferation and migration compared to overexpressing cells when tested using MTS and wound healing assays. Decreased rates of migration in wound healing assays are reversed by overexpression of the known migration-associated gene MEMO1. Additionally, cells with reduced expression of zc3h8 failed to form colonies in soft agar or pass through an extracellular matrix coating a transwell chamber- a phenotype that can be rescued using recombinant human zc3h8. Conversely, untransformed mouse mammary COMMA-1D cells gain the ability to form colonies in soft agar when transfected to stably overexpress zc3h8. Our in vitro studies suggest that zc3h8 expression has a role in tumorigenesis. These observations have been supported with in vivo studies in mice. We implanted cells knocked down for zc3h8 and control cells into the mammary glands of syngeneic female mice. Mice injected with cells that have reduced expression of zc3h8 form substantially smaller tumors by weight compared to control. When taken together, these data show that zc3h8 has a role in the formation and progression of cancer through its association with nuclear bodies involved in transcriptional regulation and RNA modification. Citation Format: John A. Schmidt, Keith G. Danielson, Jani L. Swiatek, Emily R. Duffner, Janice E. Knepper. Association of ZC3H8 with nuclear bodies and its role in promoting tumor cell behavior in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1134.

Decorin is a leucine-rich, chondroitin/dermatan sulfate proteoglycan which binds collagen and growth factors. We have recently completed the genomic organization of human decorin and discovered two alternatively spliced leader exons, designated exon Ia and Ib, in the 5'-untranslated region. Initial analysis of the sequences upstream to these two exons showed that promoter Ia contained only two GC boxes while promoter Ib contained a CAAT and two TATA boxes in close proximity to the transcription start site. To determine if these 5'-flanking sequences exhibited promoter activity, chimeric chloramphenicol acetyltransferase expression plasmids containing the promoter region of either exon Ia or Ib were transfected into HeLa and MG-63 osteosarcoma cells. The results showed that only the region flanking exon Ib was functional. In vitro transcription assay generated two transcripts of 92 and 82 base pairs (bp) indicating that both TATA boxes could be used. Using stepwise 5' deletion analysis we found that the minimum promoter region at -140 bp from the transcription start site, which contained only the CAAT and the two TATA boxes, exhibited strong promoter activity. When a larger construct containing an additional 800 bp of upstream region was tested, a significant increase in transcriptional activity was observed. Interestingly, this promoter region contained several putative binding sites for ubiquitous factors (AP1, AP5, and NF-kappa B) and for transforming growth factor-beta and a 150-bp homopurine/homopyrimidine element with several mirror repeats. When contained in a supercoiled plasmid, this sequence exhibited sensitivity to endonuclease S1, an enzyme that preferentially digests single-stranded DNA. Precise S1 mapping, obtained by direct sequencing of nine distinct S1-generated clones, revealed that in all cases the borders of the sensitive sequence resided within the pur/pyr segment. We propose that this region of the promoter could adopt an intramolecular hairpin triplex structure in vivo and may play a role in the chromatin organization at the decorin gene locus. In addition, this region was able to up-regulate a minimal heterologous promoter in transient transfection assays. The results show that the structure of the decorin gene promoter is different from that of any other proteoglycan promoter characterized so far and indicate that the pur/pyr segment plays a role in the regulation of gene transcription.

Decorin is a chondroitin/dermatan sulfate proteoglycan expressed by most vascular and avascular connective tissues and, because of its ability to interact with collagen and growth factors, has been implicated in the control of matrix assembly and cellular growth. To understand the molecular mechanisms involved in regulating its tissue expression, we have isolated a number of genomic clones encoding the complete decorin gene. The human decorin gene spans over 38 kb of continuous DNA sequence and contains eight exons and very large introns, two of which are 5.4 and > 13.2 kb. We have discovered two alternatively spliced leader exons, exons Ia and Ib, in the 5[prime] untranslated region. These exons were identified by cloning and sequencing cDNAs obtained by polymerase chain reaction amplification of a fibroblast cDNA library. Using Northern blotting or reverse transcriptase PCR, we detected the two leader exons in a variety of mRNAs isolated from human cell lines and tissues. Interestingly, sequences highly (74-87%) homologous to exons Ia and lb are found in the 5[prime]untranslated region of avian and bovine decorin, respectively. This high degree of conservation among species suggests regulatory functions for these leader exons. In the 3' untranslated region there are several polyadenylation sites, andmore » at least two of these sites could give rise to the transcripts of [approx]1.6 and [approx]1.9 kb, typically detected in a variety of tissues and cells. Using a genomic clone as the labeled probe and in situ hybridization of human metaphase chromosomes, we have mapped the decorin gene to the discrete region of human chromosome 12q23. This sturdy provides the molecular basis for discerning the transcriptional control of the decorin gene and offers the opportunity to investigate genetic disorders linked to this important human gene. 57 refs., 11 figs., 3 tabs.« less

The regulation of galectin-3 expression in skeletal tissues is not completely understood. Our studies indicate that HIF-1α regulates galectin-3 expression by interacting with hypoxia regulatory elements in the promoter region. Finally, we show that galectin-3 serves a prosurvival role in the intervertebral disc. Introduction: Earlier reports indicated that galectin-3 (gal-3) is highly expressed in the epiphyseal growth plate cartilage and the intervertebral disc. Because these skeletal tissues have a limited vascular supply and the cells reside in a low O2 environment, we determined if the oxemic status modulates gal-3 expression. Materials and Methods: Cells were cultured in normoxia (21% O2) or hypoxia (2% O2), and gal-3 expression and promoter activity were evaluated. Interaction of hypoxia inducible factor (HIF)-1α with the gal-3 promoter was confirmed by gel shift and site-directed mutagenesis. Results: There was minimal oxygen-dependent change in HIF-1α levels and no change in gal-3 expression and promoter activity in nucleus pulposus cells. In contrast, hypoxia induced gal-3 mRNA, protein, and promoter activity in HeLa cells and mouse embryonic fibroblasts (MEFs) from HIF-1α wildtype but not HIF-1–null mice. To evaluate the importance of HIF-1 in regulation of gal-3 expression, we overexpressed HIF-1α or constitutively active-HIF-1α in null MEF. An increase in gal-3 promoter activity was observed in both normoxia and hypoxia. Similarly, suppression of HIF-1α in nucleus pulposus cells, and wildtype MEF, using siRNA and pharmacological inhibitors resulted in suppression of gal-3 promoter activity and mRNA levels. Analysis of the gal-3 promoter indicated that it contained two hypoxia response elements (HREs). Gel-shift and chromatin immunoprecipitation analysis confirmed that there was binding of HIF-1α to the gal-3 HRE. Furthermore, site-directed mutagenesis of HRE completely blocked hypoxic induction of gal-3 promoter activity. In nucleus pulposus cells, suppression of gal-3 expression promoted FasL-mediated apoptosis. Conclusions: Together, these studies showed that gal-3 is a HIF-1–regulated lectin that plays an important role in nucleus pulposus cell survival.

Long-term stability of arthroplasty prosthesis depends on the integration between osseous tissue and the implant biomaterial. Integrity of the osseous tissue requires the contribution of mesenchymal stem cells and their continuous differentiation into an osteoblastic phenotype. This study aims to investigate the hypothesis that exposure to wear debris particles derived from orthopaedic biomaterials affects the osteoblastic differentiation of human mesenchymal stem cells (hMSC). Upon in vitro culture in the presence of osteogenic supplements (OS), we observe that cultures of hMSCs isolated from femoral head bone marrow are capable of osteogenic differentiation, expressing alkaline phosphatase, osteocalcin, and bone sialoprotein (BSP), in addition to producing collagen type I and BSP accompanied by extracellular matrix mineralization. Exposure of OS-treated hMSCs to submicron commercially pure titanium (cpTi) particles suppresses BSP gene expression, reduces collagen type I and BSP production, decreases cellular proliferation and viability, and inhibits matrix mineralization. In comparison, exposure to zirconium oxide (ZrO2) particles of similar size did not alter osteoblastic gene expression and resulted in only a moderate decrease in cellular proliferation and mineralization. Confocal imaging of cpTi-treated hMSC cultures revealed patchy groups of cells displaying disorganized cytoskeletal architecture and low levels of extracellular BSP. These in vitro findings suggest that chronic exposure of marrow cells to titanium wear debris in vivo may contribute to decreased bone formation at the bone/implant interface by reducing the population of viable hMSCs and compromising their differentiation into functional osteoblasts. Understanding the nature of hMSC bioreactivity to orthopaedic wear debris should provide additional insights into mechanisms underlying aseptic loosening.

Fetal liver zinc finger protein (FLIZ1, ZC3H8) was previously described as a negative regulator of GATA3 expression in T cells. GATA3 also is an important determinant of mammary cell fate and breast tumor cell behavior. We hypothesized that FLIZ1 also regulates GATA3 in the mammary gland, thereby influencing normal and tumor cell processes controlled by GATA3. We identified tumor cell lines with high levels of FLIZ1 expression, and transfected them with vectors driving expression of FLIZ1-interfering RNAs. Stable transfectants (Flizout cells) were evaluated for ability to migrate in a wound healing assay, and were found to have decreased mobility. Flizout cells also grew more slowly and with more pronounced epithelial morphology in media containing charcoal-stripped serum, suggesting increased dependence on steroid hormones. Flizout cells were compared to control cells in proliferation assays, and were found to grow at equivalent rates in standard media, but Flizout cells grew at decreased rates in the presence of tamoxifen. We implanted Flizout cells and controls into the mammary glands of syngeneic female mice, which were then treated with tamoxifen or peanut oil vehicle alone. Flizout cells grew more slowly in tamoxifen-treated animals, and tissue weights were significantly less than controls. In order to determine if FLIZ1 plays a role in the normal mammary gland, we assessed its expression during stages of mouse mammary gland development by immunoblot, and found FLIZ1 expression increased during mammary involution. These data are consistent with a role for FLIZ1 in regulation of mammary cell growth and tumor cell behavior. Citation Format: Torri Anderson, Christopher Cali, Katherine Bell, Keith G. Danielson, Ashley Klein, Christina Martin, Sara Radecki, Adam Santoro, John A. Schmidt, Janice E. Knepper. Alteration of mammary tumor cell behavior by FLIZ1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3391. doi:10.1158/1538-7445.AM2014-3391

Thrombospondin (TSP) 2, and its close relative TSP1, are extracellular proteins whose functions are complex, poorly understood, and controversial. In an attempt to determine the function of TSP2, we disrupted the Thbs2 gene by homologous recombination in embryonic stem cells, and generated TSP2-null mice by blastocyst injection and appropriate breeding of mutant animals. Thbs2−/− mice were produced with the expected Mendelian frequency, appeared overtly normal, and were fertile. However, on closer examination, these mice displayed a wide variety of abnormalities. Collagen fiber patterns in skin were disordered, and abnormally large fibrils with irregular contours were observed by electron microscopy in both skin and tendon. As a functional correlate of these findings, the skin was fragile and had reduced tensile strength, and the tail was unusually flexible. Mutant skin fibroblasts were defective in attachment to a substratum. An increase in total density and in cortical thickness of long bones was documented by histology and quantitative computer tomography. Mutant mice also manifested an abnormal bleeding time, and histologic surveys of mouse tissues, stained with an antibody to von Willebrand factor, showed a significant increase in blood vessels. The basis for the unusual phenotype of the TSP2-null mouse could derive from the structural role that TSP2 might play in collagen fibrillogenesis in skin and tendon. However, it seems likely that some of the diverse manifestations of this genetic disorder result from the ability of TSP2 to modulate the cell surface properties of mesenchymal cells, and thus, to affect cell functions such as adhesion and migration.

We report the complete genomic organization of the human WNT-5A gene, which encodes a cysteine-rich growth factor involved in cell-cell signaling during growth and differentiation. The gene comprises five exons with the terminal exon coding for a large 3'-untranslated region of approximately 6.5 kilobase pairs and utilizes multiple polyadenylation signals to generate at least four discrete transcripts. We discovered a new leader exon interrupted by a 411-base pair intron that was retained in our original cDNA cloning. The promoter region was located in a GpC-rich island and harbored numerous cis-acting elements including several GC boxes and Sp1, AP1, and AP2 binding motifs. It lacked TATA or CAAT boxes typical of housekeeping and growth factor genes. In support of this, primer extension revealed extension two transcription start sites. Transient cell transfection assays showed functional promoter activity for the 3.9-kilobase pair 5'-flanking region. Interestingly, internal and 5' deletions revealed tha the distal promoter was not required for full transcriptional activity and that the first 631 base pairs of WNT-5A harbored the strongest promoter activity. Using a panel of rodent-human hybrid DNAs carrying portions of chromosome 3p, we mapped the gene to 3p14.2-p21.1, between a constitutional and a familial renal cell carcinoma-associated translocation. In situ hybridization analyses of early human embryos at 28-42 days of gestation revealed that WNT-5A transcripts were not restricted to the developing brain and limbs but were also observed in the mesenchyme bordering the pharyngeal clefts and pouches and in the developing gonads and kidneys. The relatively high expression in the celomic epithelium and in the precursors of follicles and seminiferous tubules suggest a novel role for WNT-5A in germ-cell differentiation. This study provides the molecular basis for discerning the regulation of the WNT-5A gene and offers the opportunity to investigate genetic disorders linked to this important gene.

Biglycan is a ubiquitous chondroitin/dermatan sulfate proteoglycan that belongs to a growing family of proteins harboring leucine-rich repeats. We have cloned and sequenced the cDNA containing the complete murine biglycan, elucidated its genomic organization, and demonstrated functional promoter activity of its 5' flanking region. The deduced biglycan protein core was highly conserved across species. However, the mouse biglycan (Bgn) gene was significantly larger than the human counterpart, primarily because of a large > 4.5-kb intron between exons 1 and 2. The mouse Bgn gene spanned over 9.5 kb of continuous DNA and comprised 8 exons, with a perfectly conserved intron/exon organization vis-a-vis the human counterpart. The promoter region was enriched in GC dinucleotide and contained numerous cis-acting elements including binding sites for SP-1, AP-1 and AP-2 factors. It lacked TATA and CAAT boxes typical of housekeeping genes. In support of this, primer extension analysis showed the existence of multiple transcription start sites. Transient cell transfection assays with a construct comprising the 548 bp upstream of the major transcription start site fused to the chloramphenicol acetyl transferase reporter gene showed functional promoter activity. Internal and 5' deletion constructs showed that the distal promoter of the Bgn gene was required for full transcriptional activity. In contrast to the homologous proteoglycan decorin, the highest expression of biglycan mRNA was observed in lung, liver, and spleen of adult mouse and the lowest in skin, heart, and kidney. These results will be useful for the study of biglycan gene regulation and for the generation of mice with targeted null mutation of the Bgn gene.

Development of mammary tumors in mice carrying the retrovirus, mouse mammary tumor virus (MMTV), occurs by insertional mutagenesis in which viral transcriptional regulatory elements drive increased expression of adjacent genes. Several oncogenes have been identified due to their ability to cause loss of cell growth control in this system. However, most virus positive tumors feature multiple integration sites, and determination of which may be significant in tumor growth represents a challenge to validation of importance of integration site genes. We have developed a panel of more than 20 independent cell lines derived from MMTV-induced tumors of BALB/c mice. Analysis of common integration sites led to the identification of Tmem170, encoding a predicted transmembrane protein of 144 amino acids. Tmem170 RNA is highly expressed in several cell lines in the panel, but not in normal mouse mammary gland. Rabbit antiserum against a peptide in the predicted extracellular domain revealed a single band migrating at approximately 40 KDa in SDS gels. We developed a panel of vectors encoding silencing RNAs targeting Tmem170 and introduced these into cell lines expressing the Tmem170 RNA. Two independent cell lines exhibited decreased doubling times in culture when Tmem expression was silenced. The cells were introduced into the mammary fat pads of isogenic BALB/c mice and silenced lines exhibited dramatically decreased tumor growth. These studies support Tmem170's oncogenic role in tumor growth. In addition, the use of an easily manipulated cell line model for assessing the contribution of upregulated genes to tumorigenesis in immune competent animals allows for testing of other potential oncogenes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3874. doi:10.1158/1538-7445.AM2011-3874

Epithelial cells were isolated from mouse endometrium and cultured on two types of extracellular matrix, namely, rat-tail collagen (type I) gels and basement membrane extract (BME) derived from the Engelbreth-Holm-Swarm murine sarcoma. Cell attachment in serum-free medium during the initial 24 h after seeding was approximately twofold higher on BME compared with collagen type I. Addition of serum to the medium enhanced cell attachment on both matrices. On both collagen and BME, uterine cells grew as smooth-bordered colonies, and within a week of culture the cells became cuboidal to columnar in shape. Electron microscopy revealed the presence of apical microvilli associated with a glycocalyx, junctional complexes, tonofilaments, short strands of undilated endoplasmic reticulum, Golgi complex, and lipid droplets. However, cells on BME showed a higher degree of differentiation as assessed by occasional formation of small patches of basement membranelike structure subjacent to the flattened basal surface and formation of glandlike structures within the matrix. Proliferation of these cells as measured by radioactive thymidine incorporation into DNA was increased threefold by addition of epidermal growth factor (EGF) and insulin to the medium, but was not changed by 17 beta-estradiol. The expression of progesterone receptors by uterine epithelial cells grown on both matrices was doubled by addition of EGF and estradiol to the medium.

A cell line and subline with epithelial characteristics were established from mouse mammary hyperplastic alveolar nodules (HAN). The cells do not grow in suspension cultures in vitro or form tumors in vivo. The cells do produce significant amounts of C-type and A-type virus and low amounts of plasminogen activator.

Clonal populations were isolated from the mouse mammary cell line, COMMA-D, by transfection with a dominant-selectable gene, pSV2Neo, which confers resistance to the antibiotic, G418. Seven of twenty-four clones isolated retained the ability of the parental line to repopulate cleared mammary fat pads in vivo as ductal-alveolar hyperplasias. Two sublines designated CDNR2 and CDNR4 retained hyperplastic growth potential after multiple passages in vitro with low incidence of tumor formation. A third subpopulation, CDNR1, contained a single integration site for the pSV2Neo plasmid indicating a bonafide clonal origin for this subline. CDNR1 cells displayed heterogeneous growth phenotypes in vivo including hyperplasia, adenocarcinoma, and bone formation. Functional differentiation of CDNR1 cells organized as alveolarlike structures in vivo or on floating collagen gels in vitro was observed as determined by immunoperoxidase staining for the milk-specific protein, casein. Overall, the results indicate that a subset of cells from the COMMA-D cell line may be functionally analogous to stem cells existing in the mammary gland.

The distribution of a heavy metal binding protein, metallothionein, was studied immunocytochemically by using antimetallothionein antibody and the immunoperoxidase staining technique on histological sections of liver, kidney, intestine, lung, and testis from cadmium-treated rats. These tissues either accumulate heavy metals (e.g., liver, kidney, and testis) or are exposed to metal by ingestion or inhalation (intestine and lung). Staining for metallothionein was observed intracellularly in epithelial parenchymal cells of the liver and kidney; all hepatocytes and most renal tubular cells stained for the protein. Accumulation of metallothionein was not seen in connective tissue cells surrounding either blood vessels or renal tubules. Extracellular localization of metallothionein was also observed in the liver sinusoids and within the lumina of the renal tubules, suggesting a metal transport or excretory function for this protein. Surface columnar epithelial cells of the intestinal villi indicated the presence of metallothionein but connective tissue cells of the lamina propria were negative for the protein. The granular secretory Paneth cells of the small intestine also stained strongly for metallothionein as did respiratory epithelial cells of the lung. In the testis, metallothionein was detected in the Sertoli cells and interstitial cells but not within the spermatogonia. Sertoli cells are closely associated with the developing spermatogonia and appear to serve a nutritive role in spermatogenesis. Because of the secretory, absorptive, or nutritive function of the metallothionein-localizing cells in the organs studied, we suggest that metallothionein may be involved in metal storage or transport in addition to its commonly proposed detoxification role.