The identification of the function of genes (its biological and physiological
significance) has received considerable attention recently, expecially as a
motivating force and rallying cry for post-Human-Genome-Initiative scientific
undertakings (Fields S., 1997). However, the current pools of "functional
genomics" study designs include highly specialized model organism-based
experiments such as knockout, transgene and subsequent homology studies. In
addition, modern cell biology has provided new and efficient methods to study
in vitro, the biochemical and cellular effects of genes. Specific experiments
of functional genomics will be performed during the Centre activity on Diabetes
and Chron's disease which will be used as a model for complex diseases.
The cellular apoptotic program finely controls tissue renewal and homeostasis.
Inappropriate acceleration of the apoptotic rate often results in tissue degeneration
and functional impairment leading to disease. The genetic framework of the apoptotic
program is being revealed and mutations within a number of genes coding for
key apoptotic regulators have been shown to be pathogenetically relevant. On
the other hand, little information is still available on the status of the genes
which control of the cellular apoptotic machinery during the course of several
degenerative and inflamatory diseases, including those which the Centre will
be focusing on.
Understanding the genetics of type 2 diabetes is complicated by the fact that
multiple genes are likely to be involved in different families with type 2 diabetes
and also in a given patient with the disease. It is presently important, therefore,
both to characterize genetic defects which are known to cause diabetes (at least
in some families) and to search for new diabetes genes. Accordingly, the work
described in the present proposal is aimed to investigate a genetic defect in
the IR which causes insulin resistance in a family of NIDDM individuals, and
the potential involvement of genetic alterations in the function of the IRS-1
in NIDDM. Predictable short term benefits of these studies include the creation
of transgenic animals expressing these specific genetic abnormalities.
In Crohn's Disease (CD) gut there is an increased mucosal Th1 cell activation
and IL-12 production leading to a spontaneous "in vitro" release of
IFN-g by intestinal lamina propria mononculear cells (LPMC). In CD gut mRNA
expression for both IL-12 subunits (p35 and p40) is increased. However, IL12-p40
transcripts have not been detected in unstimulated autologous peripheral blood
mononuclear cells (PBMC) in CD and only in 1 out of 9 Ulcerative Colitis (UC)
and in 1 out of 13 control LPMC. This suggests a local modulation of IL-12 release
and that the Th1-mediated immune response differs in CD versus other inflammatory
diseases as UC. Although IL-12 signals are mediated by a high affinity receptor
(IL-12R) composed by two subunits, b1 and b2, levels of b2 subunit (IL12Rb2)
only are crucial in controlling Th1 cell development. In CD mucosa there is
an increased IL12Rb2 mRNA expression than controls, related to STAT4 phosphorilation
and associated to an increased expression of IFN-g, but not of IL-4 and IL-5.
IL-12 stimulation of normal LPMC increases the IL12Rb2 expression. These observations
indicates that upregulation of IL12Rb2 occurs in CD mucosa supporting the role
of IL-12/IL12R signaling pathway in the local immune response.
The availability of a rapidly growing number of mouse null mutants, obtained
through inactivation by homologous recombination of several genes, has represented
an invaluable source of knowledge on mammalian development, cellular biology
and physiology and has provided many models for human inherited diseases. However,
targeted ablation of genes which play essential roles during embryogenesis often
results in a lethal phenotype which impairs the trial to dissect the functional
role played by these genes in adult tissue-specific environments. This is especially
true in the case of genes encoding for molecules involved in pleiotropic signal
transduction pathways. Very likely, this will be also the case for genes involved
the transduction of insulin signals. This problem can now be overcome by the
advent of methods allowing conditional gene targeting, which opens the way for
the analysis of the consequence of a particular mutation in a defined organ
and at a specific time during the life of a mouse. Indeed, improvements of the
original 'knock-out' strategy through the exploitation of exogenous enzymatic
systems (the CRE recombinase) that are active in the recombination process,
have considerably extended the range of genetic manipulations that can be produced.
Insertion of "lox" sequences (the target of the CRE recombinase) flanking
specific coding regions of the target gene, and subsequent mating of mice carrying
the "loxed" gene with transgenic mice expressing the CRE recombinase
under the control of cell-specific promoters, allows the generation of mice
in which gene function is ablated in in selected tissues or organs. Moreover,
it is now possible to create a mouse bearing a targeted point mutation as the
unique change in its entire genome therefore allowing very fine dissection of
gene function in vivo ("knock-in" mutants). A striking example of
the informations which can be gained by such a strategy has been recently obtained
in the case of the gene encoding the c-kit receptor tyrosine kinase. Deletions
in this gene were known to cause, in the homozygous condition, a lethal phenotype
due to severe hematopoietic defects. However, the gene had been showed to be
expressed selectively also in primordial germ cells, in cells of the melanoblastic
lineages, and in adult germ cells. Signal transduction studies performed mainly
in melanoblastic and hematopoietic cell lineages (due to the absence of available
germ cell lines) had pointed to the importance of a tyrosine residue in the
intracellular portion of the receptor, which, when phosphorylated after receptor
activation by its specific ligand (Stem Cell Factor), becomes a docking site
for the p85 subunit of phosphatydilinositol 3' kinase (PI3K). Generation of
homozygous mutant mice carrying a single nucleotide substitution in the c-kit
gene, changing this tyrosine residue into phenylalanine, showed an unexpected
phenotype: mice were viable and showed no hematopoietic defect; they also showed
no pigmentation defects and a normal number of primordial germ cells in the
embryonal gonads. However, male mice (but not females) were completely sterile
due to a severe defect in spermatogonial proliferation in the post-natal testis.
Thus, a discrete point mutation in this receptor tyrosine kinase can be tolerated
in most organs requiring c-kit function, but is selectively deleterious for
spermatogenesis.
Reference
Fields S. The future is function. Nature Genet, 1997,15:325-327.
A) understanding the impact of disease-associated gene variants on the cellular
apoptotic program Products of gene variants
selected on the basis of informations provided by workpackages 1 and 2 will
be systematically evaluated for their possible ability to interfere with the
death/survival programs in human cells. Careful biochemical and functional analysis
of disease-associated gene variants will disclose their impact on tissue degeneration.
B) functional evaluation of diabetes-associated
gene variants
After the identification of messages which are differentially expressed in FDR
tissues compared to tissues from "control" subjects, the development
of the project will depend for a great part on the UNKNOWN versus the KNOWN
nature of the differentially expressed proteins. In case of known proteins,
we will establish whether their changed expression can be related to the pathogenesis
of diabetes and/or vascular complications. In the case of unknown proteins the
complete sequences have to be obtained, and the rest of the project will depend
on the type of proteins, i.e., "secreted" or "cellular"
proteins. This information will be deduced from the study of the sequence/hydrophobicity
pattern of the protein. In addition, comparison of the sequence and/or structure
with that of the characterized proteins will shed light on the function of the
proteins under study.
C) evaluation of IL-12 and IL12R in Crohn's Disease
We will assess whether DNA-sequence variations of the human IL-12p40 promoter
within human chromosome 5q31-5q33 in subgroups of CD patients is related to
an upregulation of IL-12Rb2 with subsequent increased local release of IFN-g.
The modulation of LPMC expression of IL-12Rb2 induced by corticosteroids and
anti-TNFa "in vitro" will also be tested and related to variations
of the IL-12p40 promoter. These "in vitro" observations will finally
be related to the "in vivo" responsiveness to corticosteroids and
anti-TNFa in CD.
Animal Models
The goal is the manipulation of embryonic stem cells in order to generate mice
with tissue- and/or organ-specific ablation of the genes identified in FDR relatives.
Moreover, if human variants of these new genes will be identified, the corresponding
point mutations in these genes could be introduced into mice to characterize
the functional impact of these specific mutations in the animal model. Alternatively,
we could introduce into FDR genes specific mutations which will be predicted
to alterate their function on the basis of the informations coming from biochemical
studies or from the analysis of their molecular structure.
We will also develop knockout mice to determine the role of genes identified
in WP 2 in the regulation of insulin sensitivity by peripheral tissues and insulin
secretion by pancreatic islets.