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Biomedical subjects

I J Benjamin

Publications and source records attributed to I J Benjamin.

At least 19 recordsLinked to original sources
PubMed2006

Heat shock response: lessons from mouse knockouts.

Organisms are endowed with integrated regulatory networks that transduce and amplify incoming signals into effective responses, ultimately imparting cell death and/or survival pathways. As a conserved cytoprotective mechanism from bacteria to humans, the heat shock response has been established as a paradigm for inducible gene expression, stimulating the interests of biologists and clinicians alike to tackle fundamental questions related to the molecular switches, lineage-specific requirements, unique and/or redundant roles, and even efforts to harness the response therapeutically. Gene targeting studies in mice confirm HSF1 as a master regulator required for cell growth, embryonic development, and reproduction. For example, sterility of Hsf1-null female but not null male mice established strict requirements for maternal HSF1 expression in the oocyte. Yet Hsf2 knockouts by three independent laboratories have not fully clarified the role of mammalian HSF2 for normal development, fertility, and postnatal neuronal function. In contrast, Hsf4 knockouts have provided a consistent demonstration for HSF4's critical role during lens formation. In the future, molecular analysis of HSF knockout mice will bring new insights to HSF interactions, foster better understanding of gene regulation at the genome level, lead to a better integration of the HSF pathway in life beyond heat shock, the classical laboratory challenge.

Animals↗
PubMed2001

Heat shock factor 1-mediated thermotolerance prevents cell death and results in G2/M cell cycle arrest.

Mammalian cells respond to environmental stress by activating heat shock transcription factors (eg, Hsf1) that regulate increased synthesis of heat shock proteins (Hsps). Hsps prevent the disruption of normal cellular mitosis, meiosis, or differentiation by environmental stressors. To further characterize this stress response, transformed wild-type Hsf1+/+ and mutant Hsf1-/- mouse embryonic fibroblasts (MEFs) were exposed to (1) lethal heat (45 degrees C, 60 minutes), (2) conditioning heat (43 degrees C, 30 minutes), or (3) conditioning followed by lethal heat. Western blot analysis demonstrated that only Hsf1+/+ MEFs expressed inducible Hsp70s and Hsp25 following conditioning or conditioning and lethal heat. Exposure of either Hsf1+/+ or Hsf1-/- MEFs to lethal heat resulted in cell death. However, if conditioning heat was applied 6 hours before lethal heat, more than 85% of Hsf1+/+ MEFs survived, and cells in G2/M transiently increased 3-fold. In contrast, conditioned Hsf1-/- MEFs neither survived lethal heat nor exhibited this G2/M accumulation. Coinfection with adenoviral Hsp70 and Hsp25 constructs did not fully recreate thermotolerance in either Hsf1+/+ or Hsf1-/- MEFs, indicating other Hsf1-mediated gene expression is required for complete thermotolerance. These results demonstrate the necessity of Hsf1-mediated gene expression for thermotolerance and the involvement of cell cycle regulation, particularly the G2/M transition, in this thermotolerant response.

Animals↗
PubMed2001

Matrix metalloproteinases: from biology to therapeutic strategies in cardiovascular disease.

In this comprehensive review of matrix remodeling, one central theme that bears re-emphasis is the extensivecross-talk and dynamic interactions that exist between terminally differentiated, postmitotic cells, proliferative cells, and the ECM of the cardiovascular system. The activities of MMPs and TIMPs constitute a well-orchestrated contest to maintain tissue integrity and homeostasis. Overexpression of MMPs tilts the balance in favor of irreversible tissue destruction of joints (eg, as in rheumatic disease), and efforts to curtail such errant pathways are ongoing (123). Thrombolytic therapy and percutaneous transluminal coronary angioplasty represent effective strategies for restoring antegrade flow in occluded vessels, but multiple factors preclude most patients with AMI from receiving either of these treatments. Tissue healing and remodeling is a process in which the biology of MMPs becomes universally applicable. Basic lessons from the biochemistry and enzymology of MMPs, combined with the mechanisms of gene expression, will undoubtedly impact the development of future therapies involving MMPs and their endogenous inhibitors. In addition, formidable challenges, ranging from bioavailability to tissue penetration and toxicity in animal models, face investigators using existing pharmacotherapeutics. For congenital diseases, such as Marfan syndrome, which primarily affects the connective tissue, future therapies may be targeted to the underlying pathobiology involving MMPs. Strategies aimed at correction of the genetic defect may be complemented by those to prevent or ameliorate fundamental imbalances in matrix turnover and deposition. The future challenge for cardiovascular medicine is to appropriately shift the pendulum, not to the exclusion of, but to the recognition of the dynamic interaction that exists between myocyte and nonmyocyte populations, which clearly affect the pathogenesis of many acquired and genetic disorders.

Animals↗
PubMed2000

Disruption of heat shock factor 1 reveals an essential role in the ubiquitin proteolytic pathway.

Inhibition of proteasome-mediated protein degradation machinery is a potent stress stimulus that causes accumulation of ubiquitinated proteins and increased expression of heat shock proteins (Hsps). Hsps play pivotal roles in homeostasis and protection in a cell, through their well-recognized properties as molecular chaperones. The inducible Hsp expression is regulated by the heat shock transcription factors (HSFs). Among mammalian HSFs, HSF1 has been shown to be important for regulation of the heat-induced stress gene expression, whereas the function of HSF2 in stress response is unclear. Recent reports have suggested that both HSF1 and HSF2 are affected during down-regulation of ubiquitin-proteasome pathway (Y. Kawazoe et al., Eur. J. Biochem. 255:356-362, 1998; A. Mathew et al., Mol. Cell. Biol. 18:5091-5098, 1998; D. Kim et al., Biochem. Biophys. Res. Commun. 254:264-268, 1999). To date, however, no unambiguous evidence has been presented as to whether a single specific HSF or multiple members of the HSF family are required for transcriptional induction of heat shock genes when proteasome activity is down-regulated. Therefore, by using loss-of-function and gain-of-function strategies, we investigated the specific roles of mammalian HSFs in regulation of the ubiquitin-proteasome-mediated stress response. Here we demonstrate that HSF1, but not HSF2, is essential and sufficient for up-regulation of Hsp70 expression during down-regulation of the ubiquitin proteolytic pathway. We propose that specificity of HSF1 could be an important therapeutic target during disease pathogenesis associated with abnormal ubiquitin-dependent proteasome function.

Cysteine Endopeptidases↗
PubMed2000

Induction of a heat shock factor 1-dependent stress response alters the cytotoxic activity of hsp90-binding agents.

In addition to its classic role in the cellular stress response, heat shock protein 90 (Hsp90) plays a critical but less well appreciated role in regulating signal transduction pathways that control cell growth and survival under basal, nonstress conditions. Over the past 5 years, the antitumor antibiotics geldanamycin and radicicol have become recognized as selective Hsp90-binding agents (HBA) with a novel ability to alter the activity of many of the receptors, kinases, and transcription factors involved in these cancer-associated pathways. As a consequence of their interaction with Hsp90, however, these agents also induce a marked cellular heat shock response. To study the mechanism of this response and assess its relevance to the anticancer action of the HBA, we verified that the compounds could activate a reporter construct containing consensus binding sites for heat shock factor 1 (HSF1), the major transcriptional regulator of the vertebrate heat shock response. We then used transformed fibroblasts derived from HSF1 knock-out mice to show that unlike conventional chemotherapeutics, HBA increased the synthesis and cellular levels of heat shock proteins in an HSF1-dependent manner. Compared with transformed fibroblasts derived from wild-type mice, HSF1 knock-out cells were significantly more sensitive to the cytotoxic effects of HBA but not to doxorubicin or cisplatin. Consistent with these in vitro data, we found that systemic administration of an HBA led to marked increases in the level of Hsp72 in both normal mouse tissues and human tumor xenografts. We conclude that HBA are useful probes for studying molecular mechanisms regulating the heat shock response both in cells and in whole animals. Moreover, induction of the heat shock response by HBA will be an important consideration in the clinical application of these drugs, both in terms of modulating their cytotoxic activity as well as monitoring their biological activity in individual patients.

3T3 Cells↗
PubMed1999

HSF1 is required for extra-embryonic development, postnatal growth and protection during inflammatory responses in mice.

HSF1 is the major heat shock transcriptional factor that binds heat shock element (HSE) in the promoter of heat shock proteins (Hsps) and controls rapid Hsp induction in cells subjected to various environmental stresses. Although at least four members of the vertebrate HSF family have been described, details of their individual physiological roles remain relatively obscure. To assess whether HSF1 exhibited redundant or unique in vivo functions, we created Hsf1(-/-) deficient mice. We demonstrate that homozygous Hsf1(-/-) mice can survive to adulthood but exhibit multiple phenotypes including: defects of the chorioallantoic placenta and prenatal lethality; growth retardation; female infertility; elimination of the 'classical' heat shock response; and exaggerated tumor necrosis factor alpha production resulting in increased mortality after endotoxin challenge. Because basal Hsp expression is not altered appreciably by the HSF1 null mutation, our findings suggest that this factor, like Drosophila Hsf protein, might be involved in regulating other important genes or signaling pathways. Our results establish direct causal effects for the HSF1 transactivator in regulating critical physiological events during extra-embryonic development and under pathological conditions such as sepsis to modulate pro-inflammatory responses, indicating that these pathways have clinical importance as therapeutic targets in humans.

Animals↗
PubMed1998

Stress (heat shock) proteins: molecular chaperones in cardiovascular biology and disease.

How a cell responds to stress is a central problem in cardiovascular biology. Diverse physiological stresses (eg, heat, hemodynamics, mutant proteins, and oxidative injury) produce multiple changes in a cell that ultimately affect protein structures and function. Cells from different phyla initiate a cascade of events that engage essential proteins, the molecular chaperones, in decisions to repair or degrade damaged proteins as a defense strategy to ensure survival. Accumulative evidence indicates that molecular chaperones such as the heat shock family of stress proteins (HSPs) actively participate in an array of cellular processes, including cytoprotection. The versatility of the ubiquitous HSP family is further enhanced by stress-inducible regulatory networks, both at the transcriptional and posttranscriptional levels. In the present review, we discuss the regulation and function of HSP chaperones and their clinical significance in conditions such as cardiac hypertrophy, vascular wall injury, cardiac surgery, ischemic preconditioning, aging, and, conceivably, mutations in genes encoding contractile proteins and ion channels.

Adaptation, Physiological↗
PubMed1998

Targeted disruption of heat shock transcription factor 1 abolishes thermotolerance and protection against heat-inducible apoptosis.

Heat shock transcription factor 1 (HSF1) is a member of the vertebrate HSF family that regulates stress-inducible synthesis of heat shock proteins (HSPs). Although the synthesis of the constitutively expressed and inducible members of the heat shock family of stress proteins correlates with increased cellular protection, their relative contributions in acquired cellular resistance or "thermotolerance" in mammalian cells is presently unknown. We report here that constitutive expression of multiple HSPs in cultured embryonic cells was unaffected by disruption of the murine HSF1 gene. In contrast, thermotolerance was not attainable in hsf1(-/-) cells, and this response was required for protection against heat-induced apoptosis. We conclude that 1) constitutive and inducibly expressed HSPs exhibit distinct physiological functions for cellular maintenance and adaptation, respectively, and 2) other mammalian HSFs or distinct evolutionarily conserved stress response pathways do not compensate for HSF1 in the physiological response to heat shock.

Alleles↗
PubMed1997

Temporospatial expression of the small HSP/alpha B-crystallin in cardiac and skeletal muscle during mouse development.

Although the small (22 Kd) heat shock protein/alpha B-crystallin functions as a major structural protein and molecular chaperone in the vertebrate lens, little is known about the protein's role in nonlenticular tissues such as the heart and skeletal muscle. Recent studies have demonstrated that alpha B-crystallin expression is uniquely regulated during myogenesis in vitro. We report here for the first time that the temporal and spatial expression of alpha B-crystallin is similarly regulated in vivo during mouse embryogenesis. Expression of alpha B-crystallin mRNA was detected by in situ hybridization in the primitive heart at 8.5 days postconception (p.c.) and in the myotome of the somites at 10.5 days p.c. This tissue-restricted pattern was corroborated by immunohistochemical studies. alpha B-crystallin mRNA and protein expression were uniform in the developing atria and ventricles without regional differences or gradients. alpha B-crystallin expression was absent in the endocardial cushion, pulmonary trunk, aorta, and endothelium. Examination of muscle precursors revealed expression throughout the dorsoventral aspect of the myotomes and in developing skeletal muscle. Our findings suggest that alpha B-crystallin may serve pivotal roles as a structural protein and a molecular chaperone in myofiber stabilization of metabolically active tissues during early embryogenesis. Thus, early alpha B-crystallin expression in myogenic lineages supports the hypothesis that the putative functions of alpha B-crystallin are coupled to the activation of genetic programs responsible for myogenic differentiation and cardiac morphogenesis.

Animals↗
PubMed1996

Differential expression of B-crystallin and Hsp27 in skeletal muscle during continuous contractile activity. Relationship to myogenic regulatory factors.

AlphaB-crystallin (alphaBC) is a major structural protein (22 kDa) of the ocular lens as well as a bona fide heat shock protein in non-lens tissue. The alphaBC gene is abundantly expressed in tissues with high oxidative capacity, including the heart and type I skeletal muscle fibers, and is regulated by the MyoD family of basic helix-loop-helix transcription factors during myogenesis. To test the hypothesis that alphaBC expression may be directly regulated by the demand for oxidative metabolism, we examined the expression of alphaBC and the ancestral-related Hsp27 in rabbit tibialis anterior muscle subjected to continuous low frequency motor nerve stimulation (3 V, 10 Hz). alphaBC mRNA and protein increased within the 1st day of continuous contractile activity (5- and 2.5-fold, respectively) and achieved maximum levels (>20-and 4-fold, respectively) after 21 d of stimulation. Hsp27 mRNA and protein levels also increased with stimulation, but with a less specific and dramatic induction pattern. In agreement with the Northern analysis, in situ hybridization performed on cross sections from tibialis anterior muscle revealed progressively increasing alphaBC transcript signal, localized in a ringlet pattern, from 1 through 21 days of stimulation. Serial sections subjected to myosin immunohistochemistry revealed that alphaBC expression was confined to slow-twitch type I and a subpopulation of fast twitch type II fibers after 1 day but present in nearly all fibers after 21 days of stimulation. Transcript levels of all four myogenic regulatory factors (MyoD, myogenin, myf-5, and MRF4) also increased with stimulation in a pattern temporally similar with alphaBC, suggesting that expression of alphaBC in response to stimulation may, in part, be regulated through myogenic regulatory factor(s) interaction with the canonical E-box element located within the alphaBC promotor. These data demonstrate that expression of the small heat shock protein, alphaBC, is rapidly induced independent of the ancestrally related Hsp27 in a fiber type specific pattern in skeletal muscle subjected to the oxidative stress imposed by continuous contractile activity.

Animals↗
PubMed1996

Cardioprotective effects of 70-kDa heat shock protein in transgenic mice.

Heat shock proteins are proposed to limit injury resulting from diverse environmental stresses, but direct metabolic evidence for such a cytoprotective function in vertebrates has been largely limited to studies of cultured cells. We generated lines of transgenic mice to express human 70-kDa heat shock protein constitutively in the myocardium. Hearts isolated from these animals demonstrated enhanced recovery of high energy phosphate stores and correction of metabolic acidosis following brief periods of global ischemia sufficient to induce sustained abnormalities of these variables in hearts from nontransgenic littermates. These data demonstrate a direct cardioprotective effect of 70-kDa heat shock protein to enhance postischemic recovery of the intact heart.

Adenine Nucleotides↗
PubMed1996

Continuous contractile activity induces fiber type specific expression of HSP70 in skeletal muscle.

Continuous contractile activity of skeletal muscle elicits an early and dramatic increase in ribosomal RNA, suggesting that translational efficiency and/or capacity is enhanced during the adaptive response to increased metabolic demand. In view of the important role heat shock or stress proteins (HSPs) play as molecular chaperones during protein synthesis, we examined whether expression of the inducible 70-kDa HSP (HSP70) and/or mitochondrial 60-kDa HSP (HSP60) is altered in rabbit tibialis anterior muscle during continuous low-frequency motor nerve stimulation. Induction of the HSP70 gene was evident within 24 h after the onset of stimulation as reflected by increases in HSP70 transcription (> 20-fold) and mRNA (> 50-fold). HSP70 protein levels were significantly elevated (10- to 12-fold) after 14 and 21 days of stimulation. Mitochondrial HSP60 mRNA and protein also increased during stimulation (> 18- and > 5-fold after 21 days, respectively). In situ hybridization and immunohistochemistry coupled with myosin ATPase staining revealed that expression of HSP70 was restricted to oxidative type I and IIa fibers during the first 3 days of stimulation but shifted to primarily type II fibers after 21 days of stimulation. These findings demonstrate that induction of HSP70 during the adaptive response to chronic motor nerve stimulation proceeds from type I/IIa to type IId(x)/b fibers, suggesting that the expression of HSPs may be required to support the folding and compartmentalization of nascent proteins during the transformation process.

Animals↗
PubMed1993

Human heat shock protein 70 (hsp70) protects murine cells from injury during metabolic stress.

Expression of heat shock protein 70 (hsp70) is stimulated during ischemia, but its proposed cytoprotective function during metabolic stress has remained conjectural. We introduced a human hsp70 gene into mouse 10T1/2 cells and assessed the susceptibility of these cells to injury in response to conditions that mimic ischemia. Transiently transfected cells, in the absence of stress, expressed human hsp70 to levels equal to or greater than those induced by heat shock, as assessed by RNAse protection, immunoblot, and immunohistochemical analyses. By comparison to cells transfected with a control plasmid, cells expressing the human hsp70 transgene were resistant to injury induced by glucose deprivation and inhibition of mitochondrial respiration. These results provide direct evidence for a cytoprotective function of hsp70 during metabolic stress.

Animals↗