Eric W. Howard

Gelatinase A Activation Is Regulated by the Organization of the Polymerized Actin Cytoskeleton

Gelatinase A (GL-A) is a matrix metalloproteinase (MMP) involved in both connective tissue remodeling and tumor invasion. GL-A activation is mediated by a membrane-type MMP (MT-MMP) that cleaves the GL-A propeptide. In this study, we examined the role of the actin cytoskeleton in regulating GL-A activation and MT-MMP-1 expression. Human palmar fascia fibroblasts and human fetal lung fibroblasts were cultured on a planar substratum or within different types of collagen lattices. Fibroblasts that formed stress fibers, either on a planar substratum or within an attached collagen lattice, showed reduced GL-A activation compared with fibroblasts lacking stress fibers, within either floating or stress-released collagen lattices. To determine whether changes in the organization of the actin cytoskeleton could promote GL-A activation, fibroblasts with stress fibers were treated with cytochalasin D. Within 24 h after treatment, GL-A activation was dramatically increased. Associated with this GL-A activation was an increase in MT-MMP-1 mRNA as determined by Northern blot analysis. Treatment with nocodazole, which induced microtubule depolymerization and cell shape changes without affecting stress fibers, did not promote GL-A activation. These results suggest that the extracellular matrix and the actin cytoskeleton transduce signals that modulate GL-A activation and regulate tissue remodeling.

Differential expression of matrix metalloproteinases and their tissue- derived inhibitors in cutaneous wound repair

Wound extracellular matrix is a key regulator of cell adhesion, migration, proliferation, and differentiation during cutaneous repair. The amount and organization of normal wound extracellular matrix are determined by a dynamic balance among overall matrix synthesis, deposition, and degradation. Matrix metalloproteinases (MMPs) are one family of structurally related enzymes that have the collective ability to degrade nearly all extracellular matrix components. The MMPs are broadly categorized into collagenases, gelatinases, stromelysins, and membrane-type MMPs by their substrate specificity. The aim of this study was to characterize the temporal changes in mRNA profiles for rat collagenase [matrix metalloproteinase-1 (MMP-1)], gelatinase A (MMP-2), matrilysin (MMP-7), gelatinase B (MMP-9), and membrane type 1-MMP (MT1-MMP), as well as tissue inhibitor of metalloproteinases-1 (TIMP-1), TIMP-2, and TIMP-3 during the inflammatory, granulation, and early remodeling phases of excisional skin repair. Eight full-thickness skin wounds were made on the backs of each rat (7-mm2 wounds; 16 rats;n = 128 wounds). Two animals at a time were reanesthetized, and all eight wounds on each animal were excised at 12 and 24 hours and at 2, 3, 5, 7, 10, and 14 days after injury. Six wounds from each animal were excised for RNA isolation, whereas two wounds were excised for histology. Controls consisted of nonwounded skin from identical locations in four animals. Total RNA from each time point was isolated and relative mRNA quantitation performed by using reduced-cycle reverse transcription-polymerase chain reaction. Correct polymerase chain reaction product amplification was confirmed by probing the blotted polymerase chain reaction product with a 32P-labeled oligonucleotide specific for a given MMP or TIMP. We demonstrated that the majority of MMP and TIMP mRNA induction and peak expression coincided temporally with the well-characterized inflammatory and granulation stages of repair. In conclusion, there is a distinct pattern of MMP and TIMP expression during normal excisional wound repair.

Substrate Hydrolysis by Matrix Metalloproteinase-9*

The catalytic clefts of all matrix metalloproteinases (MMPs) have a similar architecture, raising questions about the redundancy in substrate recognition across the protein family. In the present study, an unbiased phage display strategy was applied to define the substrate recognition profile of MMP-9. Three groups of substrates were identified, each occupying a distinct set of subsites within the catalytic pocket. The most prevalent motif contains the sequence Pro-X-X-Hy-(Ser/Thr) at P3 through P2′. This sequence is similar to the MMP cleavage sites within the collagens and is homologous to substrates the have been selected for other MMPs. Despite this similarity, most of the substrates identified here are selective for MMP-9 over MMP-7 and MMP-13. This observation indicates that substrate selectivity is conferred by key subsite interactions at positions other than P3 and P1′. This study shows that MMP-9 has a unique preference for Arg at both P2 and P1, and a preference for Ser/Thr at P2′. Substrates containing the consensus MMP-9 recognition motif were used to query the protein data bases. A surprisingly limited list of putative physiologic substrates was identified. The functional implications of these proteins lead to testable hypotheses regarding physiologic substrates for MMP-9.

σ Receptors: potential medications development target for anti-cocaine agents

The ability of cocaine to interact with sigma receptors suggests a viable target for medications development. Recently, numerous novel compounds and antisense oligodeoxynucleotides targeting sigma receptors have been synthesized and shown to prevent the behavioral toxicity and psychomotor stimulant effects of cocaine in animals. Protective doses of sigma receptor antagonists have also been shown to prevent changes in gene expression that are induced by cocaine. Together, the studies provide insight and promising future directions for the development of potential medications for the treatment of cocaine addiction and overdose.

Myocardin-Related Transcription Factors-A and -B are Key Regulators of TGF-β1-Induced Fibroblast to Myofibroblast Differentiation

Myofibroblasts are contractile, smooth muscle-like cells that are characterized by the de novo expression of smooth muscle α-actin (SMαA) and normally function to assist in wound closure, but have been implicated in pathological contractures. Transforming growth factor beta-1 (TGF-β1) helps facilitate the differentiation of fibroblasts into myofibroblasts, but the exact mechanism by which this differentiation occurs, in response to TGF-β1, remains unclear. Myocardin-related transcription factors-A and -B (MRTFs, MRTF-A/B) are transcriptional co-activators that regulate the expression of smooth muscle-specific cytoskeletal proteins, including SMαA, in smooth muscle cells and fibroblasts. In this study, we demonstrate that TGF-β1 mediates myofibroblast differentiation and the expression of a contractile gene program through the actions of the MRTFs. Transient transfection of a constitutively-active MRTF-A induced an increase in the expression of SMαA and other smooth muscle-specific cytoskeletal proteins, and an increase in myofibroblast contractility, even in the absence of TGF-β1. MRTF-A/B knockdown, in TGF-β1 differentiated myofibroblasts, resulted in decreased smooth muscle-specific cytoskeletal protein expression levels and reduced contractile force generation, as well as a decrease in focal adhesion size and number. These results provide direct evidence that the MRTFs are mediators of myofibroblast differentiation in response to TGF-β1.

IDENTIFICATION AND CLONING OF THE MEMBRANE-ASSOCIATED SERINE PROTEASE, HEPSIN, FROM MOUSE PREIMPLANTATION EMBRYOS

Previous studies have suggested the existence of a membrane-associated serine protease expressed by mammalian preimplantation embryos. In this study, we have identified hepsin, a type II transmembrane serine protease, in early mouse blastocysts. Mouse hepsin was highly homologous to the previously identified human and rat cDNAs. Two isoforms, differing in their cytoplasmic domains, were detected. The tissue distribution of mouse hepsin was similar to that seen in humans, with prominent expression in liver and kidney. In mouse embryos, hepsin expression was observed in the two-cell stage, reached a maximal level at the early blastocyst stage, and decreased subsequent to blastocyst hatching. Expression of a soluble form of hepsin revealed its ability to autoactivate in a concentration-dependent manner. Catalytically inactive soluble hepsin was unable to autoactivate. These results suggest that hepsin may be the first serine protease expressed during mammalian development, making its ability to autoactivate critical to its function.

Matrix Metalloproteinase-2 Expression by Vascular Smooth Muscle Cells Is Mediated by Both Stimulatory and Inhibitory Signals in Response to Growth Factors

In response to growth factors, vascular smooth muscle cells (VSMCs) undergo a phenotypic modulation from a contractile, non-proliferative state to an activated, migratory state. This transition is characterized by changes in their gene expression profile, particularly by a significant down-regulation of contractile proteins. Platelet-derived growth factor (PDGF)-BB has long been known to initiate VSMC de-differentiation and mitogenesis. Insulin-like growth factor (IGF)-I, on the other hand, has differing effects depending on the model studied. Here, we report that both IGF-I and PDGF-BB stimulated VSMC de-differentiation of rat heart-derived SMCs in culture, although only PDGF-BB was capable of inducing proliferation. Although both PDGF-BB and IGF-I stimulation resulted in decreased smooth muscle α-actin expression and increased matrix metalloproteinase (MMP)-2 expression, the response to IGF-I was significantly more rapid. The increased MMP-2 expression in response to both growth factors was due to increased transcription rates and was dependent on the action of phosphatidylinositol 3-kinase (PI3K) and its downstream effector, Akt. Both PDGF-BB and IGF-I activated PI3K/Akt to similar degrees; however, only PDGF-BB concomitantly stimulated an inhibitory signaling pathway that antagonized the effects of Akt but did not alter the extent or duration of Akt activation. Together, these findings suggest that changes in MMP-2 expression are part of the program of VSMC phenotypic modulation and that both PDGF-BB and IGF-I, despite their different abilities to induce proliferation in this model, are capable of inducing VSMC activation.

Matrix metalloproteinases and the ontogeny of scarless repair : the other side of the wound healing balance

Early gestation mammalian fetuses possess the remarkable ability to heal cutaneous wounds in a scarless fashion. Over the past 20 years, scientists have been working to decipher the mechanisms underlying this phenomenon. Much of the research to date has focused on fetal correlates of adult wound healing that promote fibrosis and granulation tissue formation. It is important to remember, however, that wound repair consists of a balance between tissue synthesis, deposition, and degradation. Relatively little attention has been paid to this latter component of the fetal wound healing process. In this study, we examined the ontogeny of ten matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in nonwounded fetal rat skin and fibroblasts as a function of gestational age. We used a semiquantitative polymerase chain reaction protocol to analyze these important enzymes at time points that represent both the scarless and scar-forming periods of rat gestation. The enzymes evaluated were collagenase-1 (MMP-1), stromelysin-1 (MMP-3), gelatinase A (MMP-2), gelatinase B (MMP-9), membrane-type matrix metalloproteinases (MT-MMPs) 1, 2, and 3, and TIMPs 1, 2, and 3. Results demonstrated marked increases in gene expression for MMP-1, MMP-3 and MMP-9 that correlated with the onset of scar formation in nonwounded fetal skin. Similar results were noted in terms of MMP-9 gene expression in fetal fibroblasts. These results suggest that differences in the expression of these matrix metalloproteinases may have a role in the scarless wound healing phenotype observed early in fetal rat gestation. Furthermore, our data suggest that the differential expression of gelatinase B (MMP-9) may be mediated by the fetal fibroblasts themselves.

Toxoplasma gondii Activates Hypoxia-inducible Factor (HIF) by Stabilizing the HIF-1α Subunit via Type I Activin-like Receptor Kinase Receptor Signaling

Toxoplasma gondii is an intracellular protozoan parasite that can cause devastating disease in fetuses and immune-compromised individuals. We previously reported that the α subunit of the host cell transcription factor, hypoxia-inducible factor-1 (HIF-1), is up-regulated by infection and necessary for Toxoplasma growth. Under basal conditions, HIF-1α is constitutively expressed but rapidly targeted for proteasomal degradation after two proline residues are hydroxylated by a family of prolyl hydroxylases (PHDs). The PHDs are α-ketoglutarate-dependent dioxygenases that have low Km values for oxygen, making them important cellular oxygen sensors. Thus, when oxygen levels decrease, HIF-1α is not hydroxylated, and HIF-1 is activated. How Toxoplasma activates HIF-1 under normoxic conditions remains unknown. Here, we report that Toxoplasma infection increases HIF-1α stability by preventing HIF-1α prolyl hydroxylation. Infection significantly decreases PHD2 abundance, which is the key prolyl hydroxylase for regulating HIF-1α. The effects of Toxoplasma on HIF-1α abundance and prolyl hydroxylase activity require activin-like receptor kinase signaling. Finally, parasite growth is severely diminished when signaling from this family of receptors is inhibited. Together, these data indicate that PHD2 is a key host cell factor for T. gondii growth and represent a novel mechanism by which a microbial pathogen subverts host cell signaling and transcription to establish its replicative niche.

Induced antitumor immunity against DMBA‐4 metastatic mammary tumors in rats using laser immunotherapy

Induced antitumor immunity is a highly effective and long‐term cure for cancer, particularly for metastatic tumors. Laser immunotherapy was developed to induce such an immunologic response. It involves intratumoral administration of a light‐absorbing dye and a specially formulated immunoadjuvant, followed by noninvasive irradiation of a near‐infrared laser. Treatment of DMBA‐4 metastatic mammary tumors in rats with this approach has resulted in local control of primary tumors and eradication of untreated distant metastases. After laser immunotherapy, rats were resistant to tumor rechallenge and developed immunity, which could be adoptively transferred. To better understand the immunity induced in this tumor model, immunization using freeze–thaw DMBA‐4 cell lysates was performed, followed by tumor challenge 21 days later. Tumor cell lysate immunization delayed the emergence of metastases but did not provide immunity against the tumor challenge. Also performed was surgical resection of primary tumors before the observation of metastatic tumors. Removal of primary tumors was unsuccessful at changing the course of tumor progression. Tumors re‐emerged at the primary sites, and metastases developed at multiple remote sites. In contrast, tumor‐bearing rats successfully treated by laser immunotherapy experienced tumor regression and eradication and developed strong resistance to repeated challenges by tumor cells of the same type. Our results show that laser immunotherapy could have potential for the treatment of metastatic tumors by inducing tumor‐specific, long‐lasting immunity.