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M M Matzuk

Publications and source records attributed to M M Matzuk.

At least 91 records · Page 5Linked to original sources
PubMed1996

Transgenic models to study the roles of inhibins and activins in reproduction, oncogenesis, and development.

With the advent of gene targeting in pluripotent mouse embryonic stem cells, it is now possible to modify the mammalian genome to generate mutant strains of mice with precise genetic mutations. The major goal of my laboratory is to generate transgenic mice to use as physiologic models to study mammalian reproduction and development. The initial focus of our research has been to generate mice deficient in inhibins, activins, activin binding proteins (i.e., follistatin), and activin receptors (i.e., activin receptor type II) to understand their interactions and roles in the hypothalamic-pituitary-gonadal axis and mammalian development. Inhibins and activins, dimeric members of the TGF-beta superfamily, were discovered due to their role in pituitary follicle stimulating hormone homeostasis. However, these proteins have later been shown to have diverse endocrine, paracrine, and autocrine functions. Activins have been shown to mediate their signals through type I and type II serine/threonine kinase receptors. The high interspecies conservation of activins, inhibins, and activin receptors and the universal presence of activins in mammals, birds, amphibians, and fish suggest an evolutionarily conserved role of these proteins in animal development. Our initial studies have demonstrated a tumor suppressor role of inhibin in the gonads and adrenals and have also suggested a role of activins in cancer cachexia-like syndrome. To further study the gonadal tumor development and the cancer cachexia-like syndrome in these mice, we have begun to generate mice with multiple genetic alterations (e.g., mice deficient in both inhibin and Mullerian inhibiting substance). We have also generated mice deficient in other components of this complex system (e.g., activin beta A, activin receptor type II, follistatin). Analysis of these transgenic mutant models has aided our overall understanding of the critical roles these proteins play in the development of the reproductive system, in the modulation of the endocrine milieu that regulates reproductive function, and in mammalian development.

Activins↗
PubMed1995

Cloning of the mouse gonadotropin beta-subunit-encoding genes, I. Structure of the follicle-stimulating hormone beta-subunit-encoding gene.

More than 16kb of genomic sequence encompassing the mouse follicle-stimulating hormone beta subunit (FSH beta)-encoding gene was isolated from a 129SvEv mouse genomic library. Comparisons of the nucleotide and deduced amino-acid sequences to the FSH beta sequences of other mammalian species confirm an evolutionarily conserved role of this important protein in reproduction.

Amino Acid Sequence↗
PubMed1995

The asparagine-linked oligosaccharides of the human chorionic gonadotropin beta subunit facilitate correct disulfide bond pairing.

The role of asparagine (N)-linked oligosaccharide chains in intracellular folding of the human chorionic gonadotropin (hCG)-beta subunit was determined by examining the kinetics of folding in Chinese hamster ovary (CHO) cells transfected with wild-type or mutant hCG-beta genes lacking one or both of the asparagine glycosylation sites. The half-time for folding of p beta 1 into p beta 2, the rate-determining step in beta folding, was 7 min for wild-type beta but 33 min for beta lacking both N-linked glycans. The p beta 1-->p beta 2 half-time was 7.5 min in CHO cells expressing the beta subunit missing the Asn13-linked glycan and 10 min for the beta subunit missing the Asn30-linked glycan. The inefficient folding of hCG-beta lacking both N-linked glycans correlated with the slow formation of the last three disulfide bonds (i.e. disulfides 23-72, 93-100, and 26-110) to form in the hCG-beta-folding pathway. Unglycosylated hCG-beta was slowly secreted from CHO cells, and beta subunit-folding intermediates retained in cells for more than 5 h were degraded into a hCG-beta core fragment-like protein. However, coexpression of the hCG-alpha gene enhanced folding and formation of disulfide bonds 23-72, 93-100, and 26-110 of hCG-beta lacking N-linked glycans. In addition, the molecular chaperones BiP, ERp72, and ERp94, but not calnexin, were found in a complex with unglycosylated, unfolded hCG-beta and may be involved in the folding of this beta form. These data indicate that N-linked oligosaccharides assist hCG-beta subunit folding by facilitating disulfide bond formation.

Amino Acid Sequence↗
PubMed1995

Functional analysis of activins during mammalian development.

Activins are dimeric (beta A beta A; beta B beta B; beta A beta B) members of the transforming growth factor-beta superfamily. They are widely expressed during murine development, are highly conserved during vertebrate evolution, and may be involved in mesoderm induction and neurulation in Xenopus laevis and Oryzias latipes. To investigate the function of mammalian activins in vivo, we generated mice with mutations either in activin-beta A or in both activin-beta A and activin-beta B. Activin-beta A-deficient mice develop to term but die within 24 h of birth. They lack whiskers and lower incisors and have defects in their secondary palates, including cleft palate, demonstrating that activin-beta A must have a role during craniofacial development. Mice lacking both activin subunits show the defects of both individual mutants but no additional defects, indicating that there is no functional redundancy between these proteins during embryogenesis. In contrast to observations in lower vertebrates, zygotic expression of activins is not essential for mesoderm formation in mice.

Activins↗
PubMed1995

Different phenotypes for mice deficient in either activins or activin receptor type II.

Activins are believed to initiate a signal transduction cascade by binding to serine/threonine kinase receptors types I and II. Activins bind to several different receptors in vitro, but the significance of this interaction in vivo has not been confirmed. To test the function of the type II activin receptor (ActRcII) in mammalian development and reproduction, we generated a null mutation in the ActRcII gene in mice using embryonic stem cell technology. We expected ActRcII-deficient mice to phenocopy activin-deficient mice. A few ActRcII-deficient mice had skeletal and facial abnormalities reminiscent of the Pierre-Robin syndrome in humans, but most lacked these defects and developed into adults; their follicle-stimulating hormone was suppressed, and their reproductive performance was defective. These findings confirm a role of ActRcII in activin signalling in pituitary gonadotrophs. The striking lack of overlap between phenotypes of ActRcII-deficient and activin-deficient mice suggests that the ligands that signal through ActRcII during embryonic development are not activins.

Activin Receptors↗
PubMed1995

Multiple defects and perinatal death in mice deficient in follistatin.

Follistatin, an activin-binding protein and activin antagonist in vitro, can bind to heparan sulphate proteoglycans and may function in vivo to present activins to their receptors. In the mouse, follistatin messenger RNA is first detected in the deciduum (on embryonic day 5.5), and later in the developing hindbrain, somites, vibrissae, teeth, epidermis and muscle. In Xenopus laevis, overexpression of follistatin leads to induction of neural tissue. Here we use loss-of-function mutant mice to investigate the function of follistatin in mammals. We find that follistatin-deficient mice are retarded in their growth, have decreased mass of the diaphragm and intercostal muscles, shiny taut skin, skeletal defects of the hard palate and the thirteenth pair of ribs, their whisker and tooth development is abnormal, they fail to breathe, and die within hours of birth. These defects are more widespread than those seen in activin-deficient mutant mice, indicating that follistatin may modulate the actions of several members of the transforming growth factor-beta family.

Animals↗
PubMed1995

Transgenic mouse models for tumour-suppressor genes.

Tumour-suppressor genes are negative regulators of cell division and growth. Over the past decade, multiple, distinct tumour-suppressor genes have been identified and cloned. In recent years, the ability to specifically manipulate the mouse genome via overexpression, underexpression or deletion of genes using transgenic expression systems and embryonic stem cell (ES) technology has led to the identification and definition of the precise function of several tumour suppressor genes in vivo. Included in this group are mice with mutations in the p53 and retinoblastoma (Rb) genes. p53 Mutant mice are highly susceptible to tumour development and will serve as excellent models to understand the aetiology and pathology of several human cancers. In contrast to the role of the Rb gene in human retinoblastomas, mice heterozygous for a mutant Rb allele do not develop retinoblastoma, but develop pituitary tumours instead. Similar ES cell technology has been used to generate alpha-inhibin deficient mice. Inhibin-deficient mice develop gonadal and adrenal tumours with nearly 100% penetrance. These studies have identified inhibin as a novel secreted tumour suppressor. In the future, many of the unidentified functions of tumour-suppressor genes can be tested using this powerful in vivo assay system.

Animals↗
PubMed1995

Synergistic effects of inhibins and müllerian-inhibiting substance on testicular tumorigenesis.

Members of the transforming growth factor-beta (TGF-beta) superfamily regulate diverse physiological processes in multiple tissues. In particular, important roles for the inhibins and müllerian-inhibiting substance (MIS) have been demonstrated in the regulation of cell growth control both in vitro and in vivo. Inhibin-deficient male and female mice develop mixed granulosa/Sertoli cell tumors with nearly 100% penetrance. MIS-deficient male mice develop as pseudohermaphrodites with oviducts and uteri. In addition, MIS-deficient males have Leydig cell hyperplasia and, in one case, a Leydig cell tumor. To determine whether MIS could modify the development of the granulosa/Sertoli cell tumors in inhibin-deficient mice or whether inhibin could alter the development of the Leydig cell hyperplasia of MIS-deficient mice, animals deficient for both inhibins and MIS were generated. Adult inhibin/MIS-deficient male mice developed testicular tumors and large fluid-filled uteri. The accumulation of uterine fluid was due in part to an increase in estradiol secretion from the tumors and was blocked by a pure estrogen antagonist, ICI 182,780. The testes of the inhibin/MIS-deficient males developed granulosa/Sertoli cell tumors and Leydig cell neoplasia earlier, grew faster, were less hemorrhagic, and produced less estradiol than the testes of inhibin-deficient controls. These results demonstrate that inhibin and MIS synergize to influence testicular tumor development.

Animals↗
PubMed1994

Development of cancer cachexia-like syndrome and adrenal tumors in inhibin-deficient mice.

Activins and inhibins, members of the type beta transforming growth factor superfamily of growth regulatory proteins, are produced in multiple tissues and affect diverse physiologic processes. Using embryonic stem cell technology, we previously demonstrated that inhibin can function as a gonadal tumor suppressor. In this study, we show that development of gonadal tumors is rapidly followed by a cancer cachexia-like wasting syndrome. Cachectic inhibin-deficient mice develop hepatocellular necrosis around the central vein and parietal cell depletion and mucosal atrophy in the glandular stomach, are anemic, and demonstrate severe weight loss. The liver pathology is consistent with studies demonstrating an effect of elevated activins on rat hepatocytes. In inhibin-deficient mice with tumors, activins are > 10-fold elevated in the serum and are likely causing some of the cachexia symptoms. In contrast, inhibin-deficient mice gonadectomized at an early age do not develop this wasting syndrome. However, these gonadectomized, inhibin-deficient mice eventually develop adrenal cortical sex steroidogenic tumors with nearly 100% penetrance, demonstrating that inhibin is also a tumor suppressor for the adrenal gland.

Activins↗
PubMed1994

Overexpression of activin-beta A subunit mRNA is associated with decreased activin type II receptor mRNA levels in the testes of alpha-inhibin deficient mice.

Activins and inhibins are polypeptides of the transforming growth factor-beta family that participate in differentiation and growth of diverse cell types, and are involved in endocrine/paracrine regulation of the hypothalamo-pituitary-gonadal axis. Mice with alpha-inhibin subunit gene deletion develop large testicular tumors. In these animals, a 200-fold increase in testicular expression of activin beta A subunit mRNA was detected using S1-nuclease protection analysis. Northern blot analysis demonstrated that a predominant mRNA form of approximately 6.5 kb and a second minor form of 4.5 kb were overexpressed in the testes of the alpha-inhibin deficient animals. Testicular expression of the type II activin receptor was decreased 3-fold in these mice. In contrast, hypothalamic beta A and type II activin receptor mRNA levels remained unaltered. alpha-Inhibin may play a role to suppress the expression of beta A mRNA in the mammalian testes. These results demonstrate that increased expression of activin is accompanied by a tissue specific reduction in the expression of its own receptor mRNA in vivo.

Activin Receptors↗
PubMed1994

Structure of the mouse growth/differentiation factor 9 gene.

Using a mouse GDF-9 cDNA as a probe, over 20 kb of sequence encompassing the GDF-9 gene was isolated from a mouse 129SvEv genomic library. Sequence analysis of the exons, exon-intron boundaries, and 5'- and 3'-non-translated regions was used to establish the structure of the mouse GDF-9 gene. The GDF-9 gene is encoded by two exons separated by a 2.9 kb intron. Multiple putative transcription start sites are detected between 31 and 57 bp upstream of the start site of translation and a putative polyadenylation signal lies 342 bp 3' of the end of translation. Knowledge of the GDF-9 gene structure will enable us to further understand the role of GDF-9 in ovarian physiology and development.

Amino Acid Sequence↗
PubMed1994

Activin/inhibin beta B subunit gene disruption leads to defects in eyelid development and female reproduction.

Inhibins and activins are dimeric growth factors of the transforming growth factor-beta superfamily, a class of peptides that can regulate the growth and differentiation of a variety of cell types. Recently, activins have been implicated in early vertebrate development through their ability to evoke, in Xenopus embryo explants, both morphological and molecular changes characteristic of mesoderm induction. To understand these processes further, we have used homologous recombination in embryonic stem cells to create mouse strains carrying mutations in the gene encoding the activin/inhibin beta B subunit. These mice are expected to be deficient in activin B (beta B:beta B), activin AB (beta A:beta B), and inhibin B (alpha:beta B). Viable mutant animals were generated, indicating that the beta B subunit is not essential for mesoderm formation in the mouse. Mutant animals suffered, however, from distinct developmental and reproductive defects. An apparent failure of eyelid fusion during late embryonic development led to eye lesions in mutant animals. Whereas beta B-deficient males bred normally, mutant females manifested a profoundly impaired reproductive ability, characterized by perinatal lethality of their offspring. The phenotype of mutant mice suggests that activin beta B (1) plays a role in late fetal development and (2) is critical for female fecundity. In addition, we have found that expression of the related beta A subunit of activin is highly upregulated in ovaries of mutant females. Altered regulation of beta A activin in beta B-deficient mice may contribute to the mutant phenotype.

Activins↗
PubMed1994

Characterization of gonadal sex cord-stromal tumor cell lines from inhibin-alpha and p53-deficient mice: the role of activin as an autocrine growth factor.

Inhibin-alpha-deficient mutant mice have been generated by a targeted deletion of the inhibin-alpha gene through homologous recombination in murine embryonic stem cells. Essentially all of the homozygous mutants develop gonadal sex cord-stromal tumors. To investigate their endocrine and proliferative characteristics, gonadal tumor cells were maintained in vitro. Cells from inhibin-alpha-deficient mice multiplied poorly; however, cells from mice deficient in both inhibin-alpha and p53 proliferated rapidly and showed higher saturation density and plating efficiency, thus allowing the establishment of clonal tumor cell lines. Although negligible estrogen and testosterone was produced by the clonal cells, high levels of progesterone were secreted. A clonal testis tumor cell line (inhibin-alpha/p53 deficient) showed no response to exogenous FSH, human CG (hCG), or inhibin A but exhibited a 6- to 8-fold increase in progesterone production in response to forskolin treatment. The stimulatory effect of forskolin was, however, partially blocked by activin treatment. Northern blot analysis revealed inhibin beta A and beta B mRNA expression in these cells. Furthermore, Western blot analyses indicated the secretion of the beta A-subunit protein. We further tested the role of activin on tumor cell growth. Treatment with follistatin, an activin-binding protein, inhibited tumor cell replication in a dose-dependent manner. In contrast, treatment with activin A stimulated tumor cell growth by itself and partially blocked follistatin action. Incorporation of thymidine into DNA of these cells was also stimulated by activin. In addition, treatment with antiactivin A serum inhibited tumor cell replication and blocked the stimulatory action of activin on cell growth. The activin action is likely mediated by specific receptors because cross-linking of [125]activin to the 50-55 kilodalton type I and 75-80 kilodalton type II receptors was found using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Northern blot analysis also revealed follistatin mRNA expression in the tumor cells, suggesting these cells are related to granulosa cells. Our findings indicate that activin can act as an autocrine growth factor in stimulating the proliferation of gonadal tumor cell lines derived from inhibin-alpha and p53-deficient mice and inhibits progesterone production. These tumor cell lines are useful for studies on the regulation of gonadal cell proliferation and steroidogenesis as well as the signaling pathway mediating activin action.

Activins↗
PubMed1994

Identification and analysis of tumor suppressor genes using transgenic mouse models.

The identification of cloning of tumor suppressor genes has mostly relied on familial human cancer predisposition syndromes and reverse genetics. Recent advances in our abilities to manipulate endogenous genes in the mouse by applying gene targeting techniques to ES cells has allowed animal models to be generated which have the same mutations as the human syndromes. Mice lacking one or both alleles of known tumor suppressor genes have been generated to evaluate the normal function of these genes. These animals have proven to be highly susceptible to tumor development, indicating that these mice are a potent in vivo assay system for tumor suppressor genes. The initiation of gonadal tumor development in mice lacking both copies of the alpha inhibin gene proved this assay and showed that inhibin is a novel tumor suppressor. Future identification of novel tumor suppressor genes will undoubtedly also include murine ES cell/gene targeting strategies for the identification and analysis of the function of these gene products in negative growth control.

Animals↗
PubMed1992

Alpha-inhibin is a tumour-suppressor gene with gonadal specificity in mice.

The inhibins are alpha:beta heterodimeric growth factors that are members of the transforming growth factor-beta family. To understand the physiological roles of the inhibins in mammalian development and reproduction, a targeted deletion of the alpha-inhibin gene was generated by homologous recombination in mouse embryonic stem cells. Mice homozygous for the null allele (inhibin-deficient) initially develop normally but every mouse ultimately develops mixed or incompletely differentiated gonadal stromal tumours either unilaterally or bilaterally. Inhibin is thus a critical negative regulator of gonadal stromal cell proliferation and the first secreted protein identified to have tumour-suppressor activity.

Animals↗
PubMed1992

Structure of the mouse activin receptor type II gene.

Knowledge of the structures of the activin receptor genes is crucial to our understanding of the role of the activins, inhibins, and their receptors in developmental and physiological processes. The type II activin receptor (ActRc) has been shown to be a transmembrane protein with putative serine/threonine kinase activity. Using a human ActRc type II cDNA as a probe, 18 overlapping lambda clones containing portions of the mouse ActRc type II gene were isolated from a mouse 129SvE genomic library. Sequence analysis of the exons, exon-intron boundaries, and 5' and 3' non-translated regions as well as Southern blot analysis of mouse genomic DNA were used to establish the structure of the mouse ActRc type II gene. The mouse ActRc type II gene is encoded by 11 exons and spans greater than 66 kilobases. Two large introns (introns 1 and 4) contribute the majority of the gene size and are found to delineate exons which encode important domains of the activin receptor. Analysis of the 5' region of the gene reveals several putative transcription factor binding sites which may be important for the complex transcriptional regulation of this gene.

Activin Receptors↗
PubMed1992

Cloning of the human activin receptor cDNA reveals high evolutionary conservation.

The entire coding region of the human activin receptor was obtained from a human testis cDNA library. Analysis of the 1539 nucleotide (513 amino acid) sequence of the receptor reveals that there are only 83 nucleotide differences compared to the coding sequence of the mouse activin receptor. Similar to its ligands, the amino acid sequence of the activin receptor is highly conserved with only two conservative amino acid differences (Lys-39 and Val-92 in human versus Arg-39 and Ile-92 in the mouse). This high degree of conservation of the activin receptor illustrates a strong evolutionary selection and confirms that activin and its receptor play an important role in development.

Activin Receptors↗