Jeffrey H. White

Distribution of plasminogen activator in different fractions of bovine milk

The type and relative amounts of plasminogen activator (PA) in different fractions of bovine milk obtained from 15 Holstein cows were examined. Raw milk was centrifuged to separate skim milk and a somatic cell pellet. PA was mainly localized within the casein fraction, being 42 times that in the serum, and in association with somatic cells. The predominant form of PA in milk casein was isolated from SDS-PAGE gel extracts and had a molecular mass of approximately 75 kDa. Its activity was increased 4.1-fold (P < 0.01) in the presence of fibrin but was unaffected by the presence of amiloride, indicating that it was due to tissue-PA. The predominant forms of PA associated with milk somatic cells were isolated from SDS-PAGE gel extracts and had molecular masses of approximately 30 and approximately 50 kDa. The activity of both proteins was unaffected by the presence of fibrin but was dramatically reduced by the presence of amiloride, indicating that they represented urokinase-PA.

Staphylococcus aureus Stimulates Urokinase-Type Plasminogen Activator Expression by Bovine Mammary Cells

Staphylococcus aureus commonly causes bovine mastitis, but bovine strains, unlike human isolates of S. aureus, do not produce the bacterial plasminogen activator, staphylokinase. By use of bovine mammary epithelial and myoepithelial cell lines, it was found that bovine S. aureus M60 and its culture filtrates induce a 3- to 10-fold increase in urokinase-type plasminogen activator activity in mammary cell-conditioned media and cellular lysates. Furthermore, transcytosis of S. aureus M60 across a mammary epithelial cell monolayer was significantly enhanced by the addition of bovine plasminogen and inhibited by aprotinin. These findings provide evidence that S. aureus M60 can trigger superactivation of host plasminogen activator production and may then utilize the plasminogen activator-plasmin(ogen) system to facilitate tissue invasion without producing staphylokinase.

Hormonal and extracellular matrix regulation of plasminogen activator in a bovine mammary epithelial cell line

The effects of lactogenic hormones on urokinase plasminogen activator (u-PA) produced by bovine mammary epithelial cells (MAC-T) were examined. High levels of u-PA activity were detected in growth arrested cells cultured on plastic. This suggests that high levels of PA activity alone are not sufficient to induce proliferation of bovine mammary epithelial cells. Cells were cultured on various extracellular matrices: plastic, fibronectin, collagen, matrigel, and laminin. Basal levels of u-PA activity in media from MAC-T cells cultured on matrigel were 1.6-, 2-, 2- and 3.5-fold higher than that of cells cultured on plastic, fibronectin, collagen, and laminin, respectively. Insulin increased (P<0.01) mammary epithelial u-PA activity on a per cell basis, an effect observed irrespectively of the type of extracellular matrix onto which cells were cultured. Not unexpectedly, insulin-like growth factor (IGF)-I, Des(1–3) IGF-I and IGF-II increased mammary epithelial u-PA activity on a per cell basis and u-PA mRNA levels, thus, mimicking the effect of insulin. Dexamethasone suppressed (P<0.01) u-PA activity but was unable to suppress the insulin-induced increase in u-PA activity of cells cultured on various extracellular matrices. These data indicate that u-PA activity is modulated by both lactogenic hormones and the type of extracellular matrix.

Synthesis of plasminogen activator inhibitor 1 by bovine mammary epithelial and myoepithelial cell lines

Conversion of plasminogen to plasmin provides an important source of proteolytic activity in the bovine mammary gland. Plasminogen activator inhibitor 1 (PAI-1) plays a key role in limiting plasminogen activation. The PAI-1 biosynthetic capabilities of various bovine mammary cells were determined. The immortalized epithelial cell lines MAC-T and BME-UV and the myoepithelial cell line BMM-UV were used as model systems. Northern blot analysis indicated that both epithelial and myoepithelial cells contained PAI-1 mRNA. Bovine PAI-1 was encoded by a single mRNA species approximately 3.0 kb long. BME-UV cells contained 2.0-fold (P < 0.01) the PAI-1 mRNA of MAC-T or BMM-UV cells. Reverse zymography indicated that both epithelial and myoepithelial cells synthesized PAI-1 protein with a molecular mass of approximately 50 kDa.

Cell cycle-dependent fluctuation of urokinase-type plasminogen activator, its receptor, and inhibitors in cultured bovine mammary epithelial and myoepithelial cells.

Bovine mammary epithelial (BME-UV1, clone E-T and BME-UV, clone E-T2) and myoepithelial (BMM-UV, clone m-T2) cell lines were used to study the modulation of cell-associated activity of urokinase-type plasminogen activator (u-PA), as well as mRNA transcripts of u-PA, its receptor (u-PAR), and inhibitors (PAI-1 and PAI-2) during the cell cycle. After release from a growth arrest accomplished by growth factor deprivation, the length of the cell cycle was determined as 19-21 h, with G1, S, and G2+M phases of 6-7, 7-9, and 5-6 h respectively. As the cell cycle progressed, accumulated cell-associated u-PA activity increased. Maximal activity occurred at the S/G2 boundary and decreased during the G2/M phases. All cell lines tested produced plasmin-specific inhibitor(s). Accumulation of u-PA mRNA peaked 3 h after stimulation into the growth cycle for m-T2 and E-T and during 3-6 h for E-T2 cells. Maximum levels of u-PAR mRNA were observed at 3 h for the E-T cell line, 6-9 h for E-T2 cells, and 3-9 h for m-T2 cells. The cell cycle distribution of the PAI-1 mRNA was similar to that of u-PA for both epithelial cell lines, while for m-T2 cells maximal accumulation of PAI-1 mRNA was detected at 3-9 h after growth initiation. The increase of PAI-2 mRNA transcription for m-T2 and E-T cells was detected at 3-6 h. The PAI-2 mRNA in E-T2 cells was under detectable levels. The data indicate that the expression of the constituents of the PA system in bovine mammary epithelial and myoepithelial cells is not cell type-dependent but is tightly connected to the phase of the cell cycle.

Thyroid Hormone Receptor-β (TRβ) Mediates Runt-Related Transcription Factor 2 (Runx2) Expression in Thyroid Cancer Cells: A Novel Signaling Pathway in Thyroid Cancer

Dysregulation of the thyroid hormone receptor (TR)β is common in human cancers. Restoration of functional TRβ delays tumor progression in models of thyroid and breast cancers implicating TRβ as a tumor suppressor. Conversely, aberrant expression of the runt-related transcription factor 2 (Runx2) is established in the progression and metastasis of thyroid, breast, and other cancers. Silencing of Runx2 diminishes tumor invasive characteristics. With TRβ as a tumor suppressor and Runx2 as a tumor promoter, a compelling question is whether there is a functional relationship between these regulatory factors in thyroid tumorigenesis. Here, we demonstrated that these proteins are reciprocally expressed in normal and malignant thyroid cells; TRβ is high in normal cells, and Runx2 is high in malignant cells. T3 induced a time- and concentration-dependent decrease in Runx2 expression. Silencing of TRβ by small interfering RNA knockdown resulted in a corresponding increase in Runx2 and Runx2-regulated genes, indicating that TRβ levels directly impact Runx2 expression and associated epithelial to mesenchymal transition molecules. TRβ specifically bound to 3 putative thyroid hormone-response element motifs within the Runx2-P1 promoter ((-)105/(+)133) as detected by EMSA and chromatin immunoprecipitation. TRβ suppressed Runx2 transcriptional activities, thus confirming TRβ regulation of Runx2 at functional thyroid hormone-response elements. Significantly, these findings indicate that a ratio of the tumor-suppressor TRβ and tumor-promoting Runx2 may reflect tumor aggression and serve as biomarkers in biopsy tissues. The discovery of this TRβ-Runx2 signaling supports the emerging role of TRβ as a tumor suppressor and reveals a novel pathway for intervention.

Thyroid Hormone Receptor β Suppression of RUNX2 Is Mediated by Brahma-Related Gene 1–Dependent Chromatin Remodeling

Thyroid hormone receptor β (TRβ) suppresses tumor growth through regulation of gene expression, yet the associated TRβ-mediated changes in chromatin assembly are not known. The chromatin ATPase brahma-related gene 1 (BRG1; SMARCA4), a key component of chromatin-remodeling complexes, is altered in many cancers, but its role in thyroid tumorigenesis and TRβ-mediated gene expression is unknown. We previously identified the oncogene runt-related transcription factor 2 (RUNX2) as a repressive target of TRβ. Here, we report differential expression of BRG1 in nonmalignant and malignant thyroid cells concordant with TRβ. BRG1 and TRβ have similar nuclear distribution patterns and significant colocalization. BRG1 interacts with TRβ, and together, they are part of the regulatory complex at the RUNX2 promoter. Loss of BRG1 increases RUNX2 levels, whereas reintroduction of TRβ and BRG1 synergistically decreases RUNX2 expression. RUNX2 promoter accessibility corresponded to RUNX2 expression levels. Inhibition of BRG1 activity increased accessibility of the RUNX2 promoter and corresponding expression. Our results reveal a mechanism of TRβ repression of oncogenic gene expression: TRβ recruitment of BRG1 induces chromatin compaction and diminishes RUNX2 expression. Therefore, BRG1-mediated chromatin remodeling may be obligatory for TRβ transcriptional repression and tumor suppressor function in thyroid tumorigenesis.