WO1999037780A1

WO1999037780A1 - Expression of the insl4 gene in human embryonic bone tissues and applications

Info

Publication number: WO1999037780A1
Application number: PCT/FR1999/000137
Authority: WO
Inventors: Anne Laurent; Dominique Bellet
Original assignee: Institut Gustave-Roussy
Priority date: 1998-01-23
Filing date: 1999-01-22
Publication date: 1999-07-29
Also published as: JP2002500888A; CA2319076A1; FR2774095A1; FR2774095B1; EP1064380A1

Abstract

The invention concerns the locating of the expression of the insulin-like 4 (ISNL4) gene in the human embryonic bone tissues and ligaments, detection and diagnostic methods, methods for marking and detecting said tissues expressing INSL4 gene expression products as well as methods for selecting compounds capable of modulating the activity of said cells. The invention also concerns medicines for treating pathologies related to the differentiation and/or abnormal proliferation of said tissues, particularly pathologies related to the abnormal development of the cartilage and/or an abnormal ossification of the bone being formed and/or the abnormal development of interarticular ligaments.

Description

EXPRESSION OF THE INSL4 GENE IN HUMAN EMBRYONIC BONE TISSUES AND APPLICATIONS.

The present invention relates to the localization of the expression of the insulin 4 gene (INSL4) in bone tissues and embryonic ligaments in humans, methods of detection and diagnosis, methods of labeling and detection of cells of said cells. tissues expressing the expression products of the ÏNSL4 gene as well as methods of selecting compounds capable of modulating the activity of said cells. The invention also relates to medicaments intended for the treatment of pathologies linked to the differentiation and / or the abnormal proliferation of said tissues, in particular the pathologies linked to the abnormal development of cartilage and / or to an abnormal ossification of the bone in formation and / or abnormal development of the interosseous ligaments.

Insulin, IGF-I, IGF-II (“Insulin-like growth factors I and II” and relaxin belong to a family of peptide hormones with certain common structures and functions, including their influence on proliferation, development, differentiation and cell metabolism (D. Le Roith, 1997).

Insulin is well known as a pancreatic endocrine hormone that regulates energy metabolism. Insulin-IGF-I growth factors are growth promoter peptides involved in the endocrine, paracrine and autocrine regulation of cell growth and which are expressed in many tissues. IGF-II has similar properties but is expressed in greater amounts during the prenatal period and is considered to be a factor in fetal growth.

Relaxin induces a remodeling of the connective tissues in the reproductive tract and inhibits uterine contractions. The relaxin gene is expressed in the corpus luteum, the decidua, the trophoblast and the prostate (Bogie, LN et al., 1995). Its functional role in the brain, where a very important expression has been observed, remains to be elucidated. We also added to this family the Ley IL (INSL3) which are currently cloned in the form of cDNA and whose biological activity is still to be defined. Ley IL transcripts are present in Leydig cells and in other tissues such as the corpus luteum, trophoblast, fetal membranes and breast tissue (Adham, IM et al., 1993; Tashima, LS et al. , 1995).

Different studies have shown the obvious importance of these genes in regulating fetal growth and stem cell development

(V.K.M. Han et al., 1996; D.J. Hansell et al., 1991, and L.S. Tashiman et al., 1995).

IGF-I and IGF-II are expressed by most fetal and embryonic tissues and regulate cell growth and differentiation as an autocrine / paracrine factor. Site-directed mutagenesis experiments performed on genes encoding growth factors IGF-I and IGF-II have demonstrated the important application of growth factor systems in the growth process (J. Baker et al., 1993 ; TM De Chiara et al., 1990, and JP Liu et al., 1993). De Chiara et al. have demonstrated a causal relationship between a disabling IGF-II gene and a growth deficiency phenotype that becomes apparent from the sixteenth day of the embryo and persists after birth. Liu et al. observed that IGF-I (- / -) mutant mice had a birth weight of approximately 60% of their normal weight and that some of them were stillborn. This peptide family has in common structural characteristics defined by the position of different cysteines essential for the formation of a tertiary structure.

It has been shown that all members of this family bind to cell surface receptors. These receptors were identified by molecular cloning and characterized in detail for insulin and IGFs. They belong to the receptor tyrosine kinase (RTK's) superfamily which includes growth factor receptors and their oncogenic analogs such as c-erbB2 / neu (EGF receptor), c-met (hepatocyte growth factor receptor), fms (CSF-1 receptor) and trk (NGF receptor). The intracellular signal transduction pathway for these receptors is characterized by tyrosine kinase activity which produces autophosphorylation of tyrosine residues on the receptor followed by a chain of events corresponding to phosphorylations. This notably includes the activation of IRS-1 (in particular for insulin and IGFs), P13K, Shc, GBR2, Sos, Ras, Raf and the protein activating mitogenesis (MAP-kinase), a kinase culminating when the phosphorylation cascade affects different cellular processes such as transcription.

The pleiotropic physiological effects of this cascade of signals in general are the subject of intensive research.

The member polypeptides of this family are all characterized by the presence of a signal peptide, a B chain, a C junction peptide or C chain and an A chain.

For these polypeptide hormones, the terms pre-prohormone, prohormone or mature hormone are defined on the basis of the presence or absence of certain elements and their arrangement in a tertiary structure. Pre-prohormone is distinguished from prohormone by the presence of the signal peptide element. The distinction between prohormone and active mature hormone is more complex and depends on the hormone considered.

For example, in proinsulin and prorelaxin, the B and A chains are located respectively at the N- and C-terminal ends and are separated by a long C junction peptide which is cleaved during the maturation phase which results in the form active hormone.

Unlike insulin and relaxin, the C peptide of IGFs is not cleaved during the maturation phase. The mature forms of the IGFs peptides contain, in addition to the domains A, B and C, two terminal carboxyl peptides (domains D and E) which are cleaved after expression. Recently, a new gene, called INSL4, from the insulin-like family

(related insulins) has been characterized (WO 95 34653, Chassin, D. et al., 1995). This gene was cloned from an early placenta cDNA library and located on chromosome 9p24.

The INSL4 gene codes for a polypeptide of 139 amino acids called "EPIL" (insulin-like peptide of early placenta). For greater clarity in the present description, the expression EPIL polypeptide means the putative polypeptide of 139 amino acids deduced from the coding nucleic acid sequence and as defined in FIG. 1.

The deduced amino acid sequence showed, in agreement with the general organization of this family of hormones, certain structural homologies such as the presence of a signal peptide, a B and A chain and a C peptide of junction or C chain, as well as the presence of 6 cysteine residues, which must probably be involved in the formation of 3 disulfide bridges.

These documents describe the organization of the INSL4 gene which is composed of two exons and an intron. These documents also show that the mRNA transcripts of the INSL4 gene have only been detected in placental tissues and weakly in the uterus (D. Chassin et al., 1995).

The INSL4 gene which also belongs to this family could be a new member of this gene family which is involved in human development. To allow us to better understand the role of these genes in embryonic and fetal development, the study of the expression pathways of these genes at different stages of development has been a successful approach (T. Strachan et al., 1997; C Ottolenghi et al., 1997, and J. Burn et al., 1995). By studying the chronology and the localization of the expression of the INSL4 and IGF-II genes in the development of the placenta and human tissues of embryos at the start of gestation by the in situ hybridization technique, the inventors have highlighted a high level of expression of INSL4 transcripts in the human trophoblast, in particular in the syncytio-trophoblasts. Surprisingly, the inventors have also demonstrated a high level of expression of INSL4 transcripts in eight-week-old fetal tissues, but only in the interosseous ligaments and in the perichondrium.

The present invention therefore relates to an EPIL 1, EPIL 2 or EPIL 3 polypeptide encoded by the INSL4 gene, characterized in that it is expressed in fetal cells of bone tissue. In the present description, the term polypeptide also denotes a protein or a peptide.

The expression EPIL 1 polypeptide is intended to denote in the present invention, a polypeptide encoded by the INSL4 gene, consisting of a single chain comprising the regions A, B and C of global amino acid sequence chosen from the amino acid sequences included between positions 18 and 139, ends included, of the amino acid sequence defined in FIG. 1, said overall sequence comprising at least the amino acid sequence comprised between positions 24 and 139, ends included, said EPIL 1 polypeptide may further comprise a signal peptide such that the amino acid sequence of the EPIL 1 polypeptide comprising the signal peptide is the sequence between positions 1 and 139, ends included, of the amino acid sequence defined in the figure 1.

The expression EPIL 2 polypeptide is intended to denote in the present invention, a polypeptide encoded by the INSL4 gene, consisting of: a) a chain A, the amino acid sequence of which is the sequence between positions 115 and 139, ends included of the amino acid sequence defined in Figure 1; b) a chain B whose amino acid sequence is chosen from a sequence between positions 18 and 58, ends included, of the amino acid sequence defined in FIG. 1, said sequence comprising at least the sequence between positions 24 and 53, ends included; said EPIL 2 polypeptide may also comprise an amino acid sequence signal peptide chosen from a sequence between positions 1 and

23, ends included, of the amino acid sequence defined in FIG. 1, said sequence of the signal peptide comprising at least the sequence between positions 1 and 17, ends included.

The expression EPIL 3 polypeptide is intended to denote in the present invention, a polypeptide encoded by the INSL4 gene, consisting of a C chain, the amino acid sequence of which is chosen from a sequence between positions 54 and 114, ends included, of the amino acid sequence defined in Figure 1, said sequence comprising at least the sequence between positions 59 and 114, ends included.

The term “bone tissue” is intended to denote in the present description, the tissues preferably constituting the long, short or flat cartilaginous bones. The invention also relates to the polypeptides according to the invention, characterized in that the fetal cells of the bone tissue are cells of the perichondrium.

The subject of the invention is also the EPIL 1, EPIL 2 or EPIL 3 polypeptides encoded by the INSL4 gene, characterized in that they are expressed in fetal ligament cells, in particular the interosseous ligaments. The invention further comprises the EPIL 1, EPIL 2 or EPIL 3 polypeptides encoded by the INSL4 gene, characterized in that they are involved in the process of differentiation, proliferation and / or regeneration of bone tissue cells and / or ligaments.

Also part of the invention, the EPIL 1, EPIL 2 or EPIL 3 polypeptides encoded by the INSL4 gene, characterized in that they are involved in the process of cartilage development and / or ossification of the bone in training.

It should be understood that the invention does not relate to polypeptides in natural form, that is to say that they are not taken in their natural environment but that they could have been obtained by purification from natural sources , or else obtained by genetic recombination, which can be glycosylated or not, or even obtained by chemical synthesis and which can then contain unnatural or modified amino acids.

The invention relates to the use of one or more antibodies directed against a polypeptide EPIL 1, EPIL 2, EPIL 3, one of their homologs or variants or one of their fragments, for detection, identification , the location and / or the assay of an EPIL 1, EPIL 2, EPIL 3 polypeptide or one of their homologs or variants, in the bone tissue and / or in the ligaments.

The invention also relates to the use of one or more antibodies directed against a polypeptide EPIL 1, EPIL 2, EPIL 3, one of their homologs or variants, or one of their fragments for the diagnosis of pathologies related to the abnormal expression of the EPIL 1, EPIL 2 and / or EPIL 3 polypeptide in the bone tissue and / or in the ligaments.

The expression “polypeptides homologous to EPIL 1, EPIL 2 or EPIL 3” will be understood to refer to the polypeptides having, with respect to said EPIL 1, EPIL 2 or EPIL 3 polypeptides, certain modifications such as in particular a deletion, a truncation, an elongation, a fusion chimeric, and / or a mutation, in particular a point mutation. Among the homologous polypeptides, those whose amino acid sequence has at least 80%, preferably 90%, of similarity with the amino acid sequences of the polypeptides according to the invention are preferred. The expression “variant polypeptide of EPIL 1, EPIL 2 or EPIL” will be understood to mean

3, all of the mutated polypeptides possibly existing naturally, in particular in humans, and which correspond in particular to truncations, substitutions, deletions and / or additions of at least one amino acid residue. These variant polypeptides correspond in particular to an abnormal expression of the EPIL 1, EPIL 2 or EPIL 3 polypeptides.

By fragment of polypeptide EPIL 1, EPIL 2, EPIL 3, or one of their homologs or variants, is meant a polypeptide of amino acid sequence comprising at least 5 amino acids, preferably 10 amino acids and 15 amino acids consecutive of said polypeptides. Among the antibodies which can be used for said detection, identification, localization and / or said assay or for said diagnosis of pathologies, mono or polyclonal antibodies or their fragments, chimeric antibodies are preferred, characterized in that they are capable of recognizing specifically an EPIL 1, EPIL 2, EPIL 3 polypeptide, one of their homologs or variants, or one of their fragments.

Specific polyclonal antibodies can be obtained from a serum of an animal immunized against, for example:

- an EPIL 1, EPIL 2, EPIL 3 polypeptide, one of their homologs or variants, or one of their fragments, in particular produced by genetic recombination or by peptide synthesis, according to the usual procedures, from a nucleic acid sequence encoding said polypeptides or amino acid sequence of said polypeptides.

The specific monoclonal antibodies can be obtained according to the conventional hybrid culture method described by Kohler and Milstein, 1975. Said antibodies are, for example, chimeric antibodies, humanized antibodies, Fab or F (ab ') 2 fragments . According to the invention, they can also be in the form of immunoconjugates or labeled antibodies in order to obtain a detectable and / or quantifiable signal.

It is understood that the expression “several antibodies” in the present description means at least two antibodies of different specificity, that is to say capable of recognizing and binding to two different epitopes or domains of said said polypeptides. It is moreover preferred according to the invention to use at least two antibodies of different specificity for the methods, the methods and the kits of the invention which include said antibodies. In this case, it is possible, for example, to use two different antibody labels or two different development systems.

These said antibodies could thus constitute a means of immunocytochemical or immunohistochemical analysis of the expression of said polypeptides on specific sections of bone tissue and / or ligaments, for example by immunofluorescence, gold labeling, enzyme immunoconjugates. They make it possible in particular to demonstrate and quantify the normal or abnormal specific presence of said polypeptides in bone tissue and / or ligaments, which makes them useful for the identification and localization of the expression of these polypeptides, or for the diagnosis of pathologies linked to the abnormal presence of said polypeptides. More generally, said antibodies can be advantageously used in any situation where the expression of one of said polypeptides must be observed qualitatively and / or quantitatively in a biological sample of bone tissue and / or ligaments.

The specific techniques and reagents for the detection, identification, localization and / or assay of antigen-antibody complexes which can be used in the methods of the invention are well known to those skilled in the art. and are, for example, ELISA, RIA or immunofluorescence techniques which can be combined, in the case where the antibodies of the invention are not already immunoconjugated or labeled, with the appropriate reagents such as immunoconjugated antibodies, labeled with fluorcscéïnc or radiolabelled capable of specifically recognizing said antibodies, as well as chromogenic substrates specific for conjugated enzymes and control reagents of positive, negative and quantitative controls.

Thus, the invention comprises a method for the detection, identification, localization and / or specific assay of an EPIL 1, EPIL 2 and / or EPIL 3 polypeptide or a fragment thereof in a biological sample of bone tissue and / or ligament, or diagnosis of pathologies linked to the abnormal presence of said polypeptide (s) in the bone tissue and / or in the ligaments, characterized in that it comprises the following stages: a) bringing said biological sample into contact with one or more antibodies directed against said polypeptide (s); b) detection, identification, location and / or determination of the antigen-antibody complex formed.

The invention also includes a kit or kit for the detection, identification, localization and / or specific assay of an EPIL 1, EPIL 2 and / or EPIL 3 polypeptide or a fragment thereof in a biological tissue sample. bone and / or ligaments, or for the diagnosis of pathologies linked to the abnormal presence of said polypeptide (s) in said biological sample, characterized in that it comprises the following elements: a) one or more antibodies directed against said polypeptide (s) ; b) where appropriate, the reagents for constituting the medium suitable for the immunological reaction; c) where appropriate, the reagents for the detection, identification and / or determination of the antigen-antibody complexes produced by the immunological reaction.

The invention also relates to the use of an oligonucleotide probe or primer capable of hybridizing specifically with a nucleic acid sequence corresponding to the INSL4 gene, for the detection, identification, 10

localization, amplification, and / or assay of nucleic acid sequence corresponding to the INSL4 gene in bone tissue and / or in ligaments.

The invention also includes the use of an oligonucleotide probe or primer capable of hybridizing specifically with a nucleic acid sequence corresponding to the INSL4 gene, for the diagnosis of pathologies linked to the abnormal expression of the INSL4 gene in tissue bone and / or ligaments.

We mean by nucleic acid sequence corresponding to the gene

INSL4-, the nucleotide sequence of the INSL4 gene as defined in FIG. 1 or any nucleotide sequences coding for a polypeptide homologous or variant of polypeptide EPIL 1, EPIL 2 or EPIL 3, their complementary sequence, the nucleotide sequence of their corresponding RNA thus than their fragments.

Said oligonucleotide probes capable of hybridizing specifically with a nucleic acid sequence corresponding to the INSL4 gene, may be labeled or fixed on a solid support by covalent bonding or not, the techniques for labeling or fixing such probes being well known from one skilled in the art.

Specific hybridization means that the hybridization is carried out under stringency conditions, in particular under temperature and ionic strength conditions such that they allow hybridization to be maintained between two globally complementary DNA fragments. By way of illustration, high stringency conditions of the hybridization step for the purpose of defining the nucleotide sequences described above are advantageously as follows.

Hybridization is carried out at a preferred temperature of 65 ° C., in the presence of a 6 × SSC buffer, 5 × Denhardt's solution, 0.5% SDS and 100 μg / ml of DNA from salmon sperm.

1 x SSC corresponds to 0.15 M NaCl and 0.05M of sodium citrate and a solution of 1 x Denhardt corresponds to 0.02% Ficoll, 0.02% of polyvinylpyrrolidone and 0.02% of bovine serum albumin.

The washing steps can, for example, be carried out for 5 to 30 min. at 65 ° C, in 2 x SSC or 1 x SSC buffer and 0.1% SDS. 11

The high stringency hybridization conditions described above for a polynucleotide of defined size will be adapted by those skilled in the art for oligonucleotides of larger or smaller size, according to the teaching of Sambrook et al. , 1989. The oligonucleotide probes or primers, and in general the nucleic acid sequences according to the invention, will have a minimum size of 10 bases and fragments of 20 bases, and preferably 30 bases, will be preferred.

Also part of the invention are the methods of detection, identification and / or specific assay of nucleic acid sequence corresponding to the INSL4 gene in a biological sample of bone tissue and / or ligament, or of diagnosis of pathologies linked to the abnormal presence of said nucleic acid sequence in said biological sample, characterized in that they comprise the following steps: a) isolation of the DNA from said biological sample to be analyzed, or obtaining a cDNA to from TARN of said biological sample; b) specific amplification of said nucleic acid sequence using at least one primer capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene; c) qualitative and / or quantitative analysis of the amplification products. The invention also includes the methods of detection, identification and / or specific assay of nucleic acid sequence corresponding to the INSL4 gene in a biological sample of bone tissue and / or ligament, or of diagnosis of pathologies linked to the abnormal presence of said nucleic acid sequence in said biological sample, characterized in that they comprise the following steps: a) contacting an oligonucleotide probe capable of hybridizing specifically with a nucleic acid sequence corresponding to the INSL4 gene with said biological sample, the DNA contained in said biological sample having, where appropriate, previously made accessible to hybridization, under conditions allowing hybridization of said probe to the DNA contained in said biological sample; 12

b) detection, identification and / or assay of the hybrid formed between said oligonucleotide probe and the DNA of said biological sample.

The invention further relates to methods of detection, identification and / or specific assay of nucleic acid sequence corresponding to the INSL4 gene in a biological sample of bone tissue and / or ligament, or of diagnosis of pathologies linked to the abnormal presence of said nucleic acid sequence in said biological sample, characterized in that they comprise the following steps: a) contacting an oligonucleotide probe immobilized on a support and capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene, with said biological sample, the DNA of the sample, having, if necessary, been previously amplified using at least one primer capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene and / or made accessible to hybridization, under conditions allowing hybridization of said probe to the DNA of said biological sample; b) bringing the hybrid formed between said oligonucleotide probe immobilized on a support and the DNA contained in said biological sample into contact, if appropriate after removing the DNA from said biological sample which has not hybridized with said probe, with an oligonucleotide probe, in particular labeled, and capable of hybridizing specifically with a nucleic acid sequence corresponding to the INSL4 gene.

In addition, part of the present invention, the kits or kits for the detection, identification and / or specific assay of nucleic acid sequence corresponding to the INSL4 gene in a biological sample of bone tissue and / or ligament, or for the diagnosis of pathologies linked to the abnormal presence of said nucleic acid sequence in said biological sample, characterized in that they comprise the following elements: a) an oligonucleotide probe capable of hybridizing specifically with an acid sequence nucleic acid corresponding to the INSL4 gene; 13

b) where appropriate, the reagents necessary for carrying out a hybridization reaction; c) where appropriate, at least one primer capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene as well as the reagents necessary for a DNA amplification reaction.

The invention also includes kits or kits for the detection, identification and / or specific assay of nucleic acid sequence corresponding to the INSL4 gene in a biological sample of bone tissue and / or ligament, or for the diagnosis of pathologies linked to the abnormal presence of said nucleic acid sequence in said biological sample, characterized in that they comprise the following elements: a) an oligonucleotide probe, called capture probe, which can be supplied pre-immobilized on a support, and capable to specifically hybridize with a nucleic acid sequence corresponding to the INSL4 gene; b) an oligonucleotide probe, called a revelation probe, in particular labeled, and capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene; c) where appropriate, at least one primer capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene as well as the reagents necessary for a DNA amplification reaction.

Also part of the invention are the methods or kits according to the invention for the diagnosis of pathologies linked to the differentiation and / or the abnormal proliferation of bone tissue and / or ligament cells, for the diagnosis of pathologies linked to development abnormal cartilage and / or abnormal ossification of the developing bone and / or for the diagnosis of osteoporosis and / or for the diagnosis of dysplasias. Among the dysplasias, there may be mentioned in particular diaphyseal dysplasias such as diaphyseal hypoplasia or osteogenesis imperfecta or else diaphyseal hyperplasia or Engelmann disease.

In the present invention, the term “diagnosis of pathologies linked to the differentiation and / or the abnormal proliferation of bone tissue and / or ligament cells” is understood to mean, or the diagnosis of pathologies linked to the abnormal development of the 14

cartilage and / or abnormal ossification of the bone in formation or for the diagnosis of osteoporosis and / or dysplasia, not only the actual diagnosis of the pathology, but also the diagnosis or prognosis of evolution of said pathology , following for example a therapeutic treatment. Thus, according to the invention, said methods or kits can be used to evaluate the effectiveness of a therapeutic treatment of said pathology.

The techniques for specific amplification and qualitative and / or quantitative analysis of nucleic sequence which can be used in the methods of the invention are well known to those skilled in the art and are, for example, methods comprising at least one amplification step called by PCR (polymerase chain reaction) or by PCR-like of the target sequence likely to be present using at least one oligonucleotide primer of nucleic sequence as defined above . The amplified products may be treated using an appropriate restriction enzyme before detecting or assaying the targeted product, in particular by a method according to the invention.

By PCR-like will be understood to mean all the methods implementing direct or indirect reproductions of the nucleic acid sequences, or in which the labeling systems have been amplified. These techniques are of course known. In general it is the amplification of DNA by a polymerase; when the original sample is an RNA, reverse transcription should be carried out beforehand. There are currently many methods for this amplification, for example the so-called NASBA “Nucleic Acid Sequence Based Amplification” (Compton, 1991), TAS “Transcription based Amplification System” (Guatelli et al., 1990), LCR “Ligase Chain Reaction ”(Landegren et al., 1988),“ Endo Run Amplification ”(ERA),“ Cycling Probe Reaction ”(CPR), and SDA“ Strand Displacement Amplification ”(Walker et al., 1992) skilled in the art. Also known to those skilled in the art, the nucleic acid quantification techniques in which, for example, the nucleic acid to be quantified is amplified by a PCR type method and in the presence of a standard nucleic acid of the same size and quantity. known and capable of hybridizing to the same primers as the target nucleic acid. 15

The amplified fragments can be identified, for example after agarose or polyacrylamide gel electrophoresis, or after a chromatographic technique such as gel filtration or ion exchange chromatography. The specificity of the amplification can be controlled for example by molecular hybridization using as probes the probes as defined above, in particular labeled.

The invention furthermore comprises the methods of labeling a bone tissue and / or ligament cell, in particular of fetal origin, characterized in that they comprise the following steps: a) bringing a biological sample of bone and / or ligament tissue with one or more antibodies directed against the polypeptide (s) EPIL 1, EPIL 2 and / or EPIL 3, said antibodies being capable of being labeled; b) where appropriate, labeling of said antibodies using labeled compounds capable of recognizing said antibodies. Techniques for labeling cells with antibodies specific for a compound of said cell are well known to immunocytochemists or immunohistochemists and will not be developed in the present description.

Also included in the present invention are the methods of labeling a bone tissue and / or ligament cell, in particular of fetal origin, characterized in that they comprise the following steps: a) bringing a biological sample of bone and / or ligament tissue with one or more oligonucleotide probes capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene, said probes being capable of being labeled; b) where appropriate, labeling of said probes using labeled compounds capable of recognizing said probes.

The subject of the invention is also a method for detecting an abnormal cell of bone tissue and / or ligament, characterized in that it implements a labeling method according to the invention. Another aspect of the invention relates to methods of selecting a chemical or biochemical compound capable of modulating differentiation, regeneration and / or 16

proliferation of human and / or animal cells of bone tissue and / or ligament, characterized in that they use an EPIL 1, EPIL 2, EPIL 3 polypeptide, one of their homologous or variant polypeptides, or one of their fragments, a nucleic acid sequence corresponding to the INSL4 gene, or a bone or ligament cell, in particular a fetal cell. Said cell can be a cell transformed by genetic recombination or selected according to the invention.

Among these methods, preference is given to methods characterized in that said selected compound is capable of inhibiting or promoting the differentiation, regeneration and / or proliferation of human and / or animal cells of bone tissue and / or ligament, in particular the fetal cells of the perichondrium.

Said compounds may for example be selected on their capacity to bind to an EPIL 1, EPIL 2, EPIL 3 polypeptide, to one of their homologous or variant polypeptides, or to one of their fragments, and to inhibit or promote the activity of said polypeptides in bone tissue and / or in ligaments, as, for example, agonist or antagonist ligands of a receptor specific for said polypeptides on cells of said tissues.

Said compounds may also be selected on their ability to bind to a nucleic acid sequence corresponding to the INSL4 gene, and to inhibit or promote the expression of the gene coding for said polypeptides as an inducer or repressor.

Said compounds can also be selected on their capacity to modulate on said cells in vitro or in vivo, the biological activity of said polypeptides expressed by said cells, such as in particular the differentiation, proliferation or regeneration of said cells. For example, the invention includes a method of selecting compounds capable of binding to an EPIL 1, EPIL 2, EPIL 3 polypeptide, to one of their homologous or variant polypeptides, or to one of their fragments, or even capable of binding link to a nucleic acid sequence corresponding to the INSL4 gene, and capable of modulating the activity of said polypeptides in bone tissue and / or in ligaments, characterized in that it comprises the following steps: 17

a) bringing said compound into contact with said polypeptide or said nucleic acid sequence; b) determining the ability of said compound to bind with said polypeptide or said nucleic acid sequence; c) demonstration of the modulation of the activity of said polypeptides on bone tissue and / or ligament cells, capable of being obtained with the compounds selected in step b).

It will be possible, for example, to use bone tissue and / or ligament cells transformed by genetic recombination or selected according to the invention, in step b) and / or c) of the present method.

The invention also includes chemical or biochemical compounds, which can be selected by a method according to the invention.

The compounds which can be selected can be organic compounds such as polypeptides or carbohydrates or any other compounds already known, or new organic compounds developed from molecular modeling techniques and obtained by chemical or biochemical synthesis, these techniques being known to those skilled in the art.

A subject of the invention is furthermore compounds according to the invention as a medicament, preferably chosen from: a) an EPIL 1, EPIL 2 or EPIL 3 polypeptide, one of their homologous or variant polypeptides or one of their fragments, obtainable by genetic recombination or chemical synthesis; b) an antibody directed against a polypeptide as defined in a); c) a chemical or biochemical compound, characterized in that it is a ligand of a polypeptide as defined in a); d) a vector comprising all or part of a nucleic acid sequence corresponding to the INSL4 gene; e) a vector as defined in d), having on its surface a specific marker of bone tissue cells or target ligament whose activity is sought to modulate; 18

f) a sense or antisense nucleic acid sequence capable of hybridizing specifically with a nucleic acid sequence corresponding to the INSL4 gene.

The fragments of EPIL 1, EPIL 2 or EPIL 3 polypeptide, or of their homologous or variant polypeptides, and as defined in a) will preferably be characterized in that they are biologically active.

The term “biologically active fragment” is intended to denote in particular a fragment of the amino acid sequence of the EPIL 1, EPIL 2 or EPIL 3 polypeptide, or of their homologous or variant polypeptides exhibiting at least one of the activities of the said polypeptides such as in particular: - the capacity to modulate, in particular to inhibit or promote, the differentiation, the proliferation and / or the regeneration of cells of bone tissue and / or ligament; and or

- the ability to be recognized by an antibody specific for an EPIL 1, EPIL 2 or EPIL 3 polypeptide, or one of their homologous or variant polypeptides. The EPIL 1, EPIL 2 or EPIL 3 polypeptides, their homologous or variant polypeptides or their recombinant fragments may be obtained from methods well known to those skilled in the art using cloning vectors and / or d expression containing a nucleic acid sequence encoding said polypeptides and host cells transformed by said vectors. The recombinant polypeptides obtained can be both in glycosylated and non-glycosylated form and may or may not have the natural tertiary structure.

These polypeptides can be produced from the nucleic acid sequences coding for said polypeptides according to the techniques for producing recombinant polypeptides known to those skilled in the art. In this case, the nucleic acid sequence used is placed under the control of signals allowing its expression in a cellular host.

An efficient system for producing a recombinant polypeptide requires a vector, for example of plasmid or viral origin, and a compatible host cell. 19

The cellular host can be chosen from prokaryotic systems, such as bacteria, or eukaryotic systems, such as, for example, yeasts, insect or mammalian cells such as CHO cells (Chinese hamster ovary cells) or murine cells or any other system advantageously available. A preferred cellular host for the expression of the proteins of the invention is constituted by Chinese hamster ovary CHO cells, and by 3A-subE cells of human placental origin.

The vector must include a promoter, translation initiation and termination signals, as well as appropriate regions for transcription regulation. It must be able to be maintained stably in the cell and may possibly have particular signals specifying the secretion of the translated protein.

These different control signals are chosen according to the cellular host used. For this purpose, the nucleic acid sequences can be inserted into vectors with autonomous replication within the chosen host, or integrative vectors of the chosen host.

Such vectors will be prepared according to the methods commonly used by those skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods, such as for example lipofection, electroporation, heat shock.

The host cells transformed by the preceding vectors can be obtained by the introduction into these cells of a nucleotide sequence inserted into a vector as defined above, then the cultivation of said cells under conditions allowing replication and / or expression of the transfected nucleotide sequence.

Among the host cells which can be used for these purposes, it is of course possible to mention bacterial cells (Olins and Lee, 1993), but also yeast cells (Buckholz, 1993), as well as animal cells, in particular cell cultures. mammals (Edwards and Aruffo, 1993), and in particular Chinese hamster ovary (CHO) cells, human cells such as 3A-subE cells 20

of placental origin, but also insect cells in which methods using baculoviruses can be used, for example (Luckow, 1993).

The methods of purification of recombinant or synthetic polypeptide used are known to those skilled in the art. The recombinant or synthetic polypeptide can be purified using methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity techniques using specific mono or polyclonal antibodies.

A preferred variant consists in producing a recombinant polypeptide fused to a “carrier” protein (conjugated protein). The advantage of this system is that it allows stabilization and a decrease in the proteolysis of the recombinant product, an increase in the solubility during the in vitro renaturation and / or a simplification of the purification when the fusion partner has a affinity for a specific ligand.

The EPIL 1, EPIL 2 or EPIL 3 polypeptides, their homologous or variant polypeptides or their fragments can also be obtained by chemical synthesis. The polypeptides obtained by chemical synthesis may contain unnatural amino acids.

Among said compounds selected as a medicament, mention may be made in particular of antisense oligonucleotides, that is to say those whose structure ensures, by hybridization with the target sequence, an inhibition of the expression of the corresponding product. Mention should also be made of sense oligonucleotides which, by interaction with proteins involved in the regulation of expression of the INSL4 gene, will induce either an inhibition or an activation of this expression.

The invention relates to compounds according to the invention, intended for the treatment of pathologies linked to the differentiation and / or the abnormal proliferation of bone tissue and / or ligament cells, in particular of pathologies linked to the abnormal development of cartilage and / or to abnormal ossification of the developing bone, in particular intended for the treatment of osteoporosis and / or dysplasia.

Among said dysplasias, diaphyseal dysplasias such as diaphyseal hypoplasia or osteogenesis imperfecta and diaphyseal hyperplasia or Engelmann's disease are particularly preferred. 21

The invention finally relates to compounds according to the invention, intended for the treatment of pathologies involving the regeneration of bone tissue and / or ligament, in particular to treatment intended to repair lesions of bone tissue and / or ligaments following to traumatic shock. The compounds of the invention as active principles of medicament, will preferably be in soluble form, associated with a pharmaceutically acceptable vehicle.

Preferably, these compounds will be administered by the systemic route, in particular by the intravenous route, by the intramuscular, intradermal route or by the oral route.

Their optimal methods of administration, dosages and dosage forms can be determined according to the criteria generally taken into account in establishing a treatment adapted to a patient such as for example the patient's age or body weight, the severity of his general condition, tolerance to treatment and side effects observed, etc.

It is also generally possible to compensate for the deficiency or overexpression of polypeptides according to the invention by a so-called “replacement” therapy allowing the amplification or the reduction of the activities of said polypeptides.

Replacement therapy may be carried out by gene therapy, that is to say by introducing the nucleic acid sequences according to the invention and / or the corresponding genes with the elements which allow their expression in vivo, in the case where one of the genes is insufficiently expressed, for example, or when it is expressed in an abnormal form.

The principles of gene therapy are known. Viral vectors can be used, for example on the basis of adenoviruses (Perricaudet et al., 1992), AAVs, Adenovirus Associated Nirus (Carter, 1993), retroviruses (Temin, 1986), poxviruses or herpes viruses ( Epstein et al., 1992). Most of the time, these viruses are used in defective form and, in general, with or without integration into the cell genome. It is also possible to provide non-viral vectors, that is to say synthetic vectors, which mimic viral sequences or which are constituted by 22

naked DNA or RNA according to the technique developed in particular by the company VICAL.

In most cases, targeting elements ensuring specific expression will have to be provided so as to be able to limit the areas of expression of the polypeptides. It is even advantageous, in certain cases, to have vectors of transient expression or at least of controlled expression which can be blocked when necessary.

The compounds according to the invention which can be used as medicament offer a new approach for the treatment of diseases linked to the unregulated differentiation or proliferation of bone cells and / or ligaments or to the absence or insufficiency certain cells of bone tissue and / or ligaments, for example following a trauma.

Other characteristics and advantages of the invention appear in the following description with the examples and the figures, the legends of which are shown below.

Legend of Figures

Figure 1: Nucleic acid sequence of the cDNA of the INSL4 gene and amino acid sequence of the EPIL polypeptide encoded by this sequence.

Figures 2A, 2B, 2C, 2D, 2E, 2F: Comparison of the distribution of INSL4 and IGF-F mRNA in the 14-week-old human placenta;

- observation on a light background (FIG. 2A) and on a black background (FIG. 2B) of the same section of tissue hybridized with an INSL4 antisense probe;

- enlargement of the previous section observed on a light background (Figure 2C);

- observation on a light background (Figure 2D) and on a black background (Figure 2E) of the same section of tissue hybridized with an antisense IGF-II probe;

- enlargement of the previous section observed on a light background (Figure 2F); s: syncytiotrophoblast; c: cytotrophoblast; m: mesenchyme; the scale bar represents 295 μm for figures 2A, 2B, 2D and 2E, and represents 15 μm for figures 2C and 2D. Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H: Cellular distribution of IGF-II mRNA in human embryos aged 25 days (Figures 3 A and 3B), 30 days 23

(Figures 3C, 3D, 3E and 3F) or 45 days (Figures 3G and 3H) obtained by in situ hybridization using an oligonucleotide probe labeled with ³³ S.

Microphotographs on a light background (left panel) and on a black background (right panel) of the same section of tissue: - axial section (Figures 3A and 3B) at the level of the "rhombencephalon"; nt: neural tube; ov: otic vesicle;

- longitudinal abdominal section (Figures 3C and 3D); nt: neural tube; m: mesonephron;

- sagital section at the level of the gill arches (ba) (Figures 3E and 3F); - longitudinal section of the vertebral body (vb) (Figures 3G and 3H); the scale bar represents 295 μm for Figures 3A, 3B, 3C and 3D, and represents 150 μm for all the other panels.

Figures 4A, AB, 4C, 4D, 4E and 4F: In situ hybridization performed with an INSL4 oligonucleotide probe on an 8 week old human fetus. Light micrographs on a light background (left panel) and black background (right panel) of the same tissue section:

- section of the distal part of the femur in the developed hip joint (Figures 4A and 4B) at the level of the "rhombencephalon"; f: femur; 1: ligament;

- longitudinal section of the spine (Figures 4C and 4D); INSL4 mRNA is located in the inter-vertebral discs, in the intervertebral ligaments; vb: vertebral body; 1: ligament;

- rib cross section (Figure 4E and 4F); pc: perichondrium; r: coast; the scale bar represents 295 μm for figures 4A, 4B, 4C and 4D, and represents 150 μm for figures 4E and 4F.

EXAMPLES

Materials and methods

A) Experimental subjects Morphologically normal embryos three to eight weeks old

(after ovulation) were obtained after legal abortion by mifepristone (RU 486) 24

at the Broussais Hospital in Paris. Complete independence was respected between the medical management of the hospital and the research group. Maternal consent was never sought before the abortion was decided and carried out. Consent was obtained after explaining the research objective and only after medical abortion was performed. This procedure is approved by the Ethics Committee of the Hospital for Sick Children in Necker (Paris). The embryos were separated from the trophoblast by dissection under a microscope. The stage of development of each embryo was estimated using the Carnegie classification established by O'Rahilly (R. O'Rahilly et al., 1987 and 1994). The microdissected embryos were frozen and stored at -80 ° C. Cryocuts (12 μm) were mounted on pretreated slides (Probon Plus slides, Microm France). The sections were fixed for 30 minutes in a solution of para-formaldehyde in 0.1 M phosphate buffer (pH 7.4), rinsed once in PBS (saline phosphate buffer) and briefly in water, then dehydrated with a series of ethanol solutions at different concentrations (50%, 75%, 95%). The sections were then air dried and stored at -80 ° C to preserve the mRNA.

B) Hybridization probe

Oligonucleotide probes of 60-mer were synthesized and purified (DNAgency, Malvern, PA). They were labeled in 3 'with [α- ^3î S] dATP (NEN, Boston, MA) using a terminal deoxyribonucleotidyl transferase (Gibco BRL, Grand Island, NY) with a specific activity of approximately 7 × 10 ⁸ cpm / mg. The oligonucleotide probes were purified on Biospin columns (Bio-Rad Laboratories, Hercules, CA) before use. The sequence of the INSL4 antisense probe is as follows (SEQ ID No. 2):

5 '-CTGGGGTGGTGGTGAATGTCTTCTC AGGC ATGGGGC A ATATGAC AGC AA GTGTTTTCCAA-3', located in exon 1. The sequence of the antisense IGF-II probe is as follows (SEQ ID N ° 3): 5 '-AGCC AATCGATTTTGTAC ATGTTTCGTCCG AGGG-3 ', located on exon 7 in the common coding region of iGF-13 mRNAs. Sense probes were used as a control. 25

C) In situ hybridization

The experimental protocol (M. Abitbol et al., 1993, and M. Gérard et al., 1995) includes a hybridization step using a solution containing 50% formamide, 4 x SSC (standard saline citrate solution), 1 x Denhardt solution, 0.25 mg / ml yeast tRNA, 0.25 mg / ml herring sperm, 0.25 mg / ml poly-A, 10% dextran sulfate, 100 mM dithiothreitol ( DTT) and a probe labeled [- ³³ S] dATP with specific activity of 6 x 10 ^δ cpm / ml. 100 μl of the hybridization solution was deposited on each of the sections. Then the sections were covered with a parafilm and incubated in a humid chamber at 43 ° C for 20 hours. After hybridization, the slides were washed twice in 1 x SSC containing 10 mM DTT for 15 minutes at 55 °, and twice in a solution of 0.5 x SSC containing 10 mM DTT for 15 minutes at room temperature. The sections were then immersed in water, dehydrated with a series of ethanol solutions of different concentrations and then exposed for one week on X-ray films Amersham Hyperfilm Betamax and in a Kodak NTB2 photographic emulsion (Eastmman Kodak, Rochester, NY) for one to two months at 4 ° C. The sections were then developed and labeled with toluidine blue. The results are analyzed by phase contrast microscopy.

Example 1 In situ hybridization of placental and fetal membranes

In order to demonstrate the specificity of the oligonucleotide probes, a Northern blot analysis was carried out with 12 μg of total RNA from trophoblast tissues. INSL4 transcripts were detected at a size of 0.7 kilobase (kb) with both the oligonucleotide probes and a cDNA probe, in agreement with the size of the INSL4 transcript published by Chassin et al. (1995). For the IGF-II gene, a major transcript of 6.0 kilobases and other transcripts of 2 to 5 kilobases were detected, confirming the results of Han et al. (1988), who identified multiple IGF-II transcripts with estimated sizes of 1.8, 2.0, 2.8, 3.0, 4.0, 4.8 and 6.2 kb, the the latter being the most abundant. These results show the specificity of the INSL4 and IGF-II oligomers which have been chosen respectively for the human INSL4 and IGF-II mRNAs. In situ hybridization performed with sense oligonucleotides 26

INSL4 and IGF-II generate only diffuse grains corresponding to the background noise, thus demonstrating the specificity of the positive hybridization obtained with antisense oligonucleotides.

A low level of expression of the INSL4 gene was detected from the 25-day-old early placenta and an abundant level of INSL4 mRNA was identified from the 14-week-old placenta. The INSL4 gene is expressed in syncytiotrophoblasts and less abundantly in cytotrophoblasts. The cells of the central part of the villous mesoderm and the umbilical cord cells express the INSL4 gene even more weakly (FIGS. 2A, 2B and 2C). No signal was detected either in the amnion or in the yolk sac (results not shown).

IGF-II mRNA was abundantly expressed in intermediate trophoblast columns and in the villous stroma at 20 days and 14 weeks of development, but was not detectable in syncytiotrophoblast sections (Figures 2D, 2 @ and 2F). In fetal membranes (20 days), abundant amounts of IGF-II mRNA have been identified in the amnion cells and the outer cells of the yolk sac (results not shown). These results are summarized in Table I below.

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TABLE I: Distribution of INSU and IGF-II mRNA in the placenta and human fetal membranes

Cell Type INSL4 mRNA IGF-II mRNA

Placenta

Syncytiotrophoblasts + + + + Neg.

Cytotrophoblasts + + + +

Villous stroma + + + +

Intermediate Trophoblasts + + + + +

Fetal membranes

+ Nd cells Umbilical cord

Amnion Neg. + + + +

Vitellin bag Neg. + + + +

Legend of Table 1:

Nd: not determined; Neg. : negative hybridization signal; +: weak hybridization signal; + +: moderate hybridization signal; + + +: strong hybridization signal;

+ + + +: very strong hybridization signal; signals are observed after 6 weeks of exposure.

The INSL4 gene is expressed more prominently in syncytiotrophoblasts than in cytotrophoblasts. These results are confirmed by the RT-PCR technique (polymerase chain reaction amplification using reverse transcriptase) carried out on the total RNA of syncytiotrophoblast and cytotrophoblast cells (D. Bellet et al, 1997). IGF-II mRNA is significantly more abundant than INSLA mRNA in adjacent sections tested with the INSL4 oligonucleotide probe. The expression of the IGF-II gene in the placenta of a human embryo aged 25 to 40 days and 14 weeks is localized in the cells of the central part of the villous mesoderm and in the cells of 28

intermediate or villous cytotrophoblast. IGF-II transcripts are not detected in the syncytiotrophoblastic layer for the development stages studied. Similar observations have been reported for example by V.K.M. Han (V.K.M. Han et al., 1996). Thus, it is likely that these two genes play a different role in the development of the trophoblast.

Example 2 In situ Hybridization of Embryonic Tissues

No INSL4-labeled oligonucleotide probe was detected in embryonic tissues 20 or 30 days old, suggesting that the INSL4 gene is not expressed at this early stage or that the level of transcript is too low to be detected by in situ hybridization. In contrast, the IGF-II gene was strongly expressed at this early embryonic stage. The expression of the IGF-II gene has been demonstrated in several tissues derived from the mesoderm and the endoderm (Figures 3 A to 3H), results consistent with those previously described (VKM Han et al., 1987a and 1987b, JE Lee et al., 1990). The major site of VIGF-II expression is localized in the liver for all the stages studied.

The 8-week-old fetal tissue sections were also hybridized using the INSL4 oligonucleotide probes. Surprisingly, the INSL4 mRNA shows a specific spatial distribution. In fact, the majority of INSL4 transcripts are located in the perichondrium of the four limbs, ribs and vertebrae. In addition, INSLA mRNA is very abundant in the interosseous ligaments (Figures 4A to 4F). In contrast, no markings were detected in the liver, lungs, heart, thymus, stomach, muscle or intestines at this stage of development. Table II below summarizes the results in accordance with the types of cells identified in the fetal tissue sections tested. O 99/37780

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TABLE II: Distribution of INSU and IGF-II mRNA in embryonic tissues (25, 30 and 45 days old for VIGF-II and 8 weeks old for VINSL4)

INSU embryonic tissues INSU mRNA IGF-II mRNA

Méninges Neg. + +

Mandibular prominence Neg. + +

Branchial arches Neg. + +

Thymus Neg. Nd

Aortic arches Neg. +

Heart Neg. +

Bone marrow Neg. Nd

Lung Neg. +

Liver Neg. + + +

Stomach Neg. Nd

Pancreas Neg. Nd

Intestines Neg. Nd

Adrenal glands Neg. + +

Metanephros Neg. + +

Sclerotome Neg. +

Perichondrium + + + +

Ligaments + + Nd

Skeletal muscles Neg. + +

Neg skin. Neg.

Legend of Table II:

+ + + +: very strong hybridization signal; signals are observed after 6 weeks of exposure. 30

Slices of hybrid embryonic tissues with the same oligonucleotide probes of INSL4 and IGF-II show that, on the contrary, transcripts of IGF-II, which are localized in most embryonic tissues (VRK Han et al., 1987a et 1987b; JE Lee et al., 1990), the INSL4 gene is expressed only in a few of these tissues. In fact, in sections of 8-week-old human embryo tissue, INSL4 transcripts are only located in the cells of the perichondrium and ligaments. At this stage of development, chondrification is started for all the bones of the lower limbs.

It is remarkable that the INSL4 gene is a member of the insulin related genes superfamily and that other members of this family encode polypeptides that regulate both growth and development in many tissues. In particular, insulin-related growth factors (IGFs) play an important role in regulating bone formation (B. Bhaumic et al., 1993; R. Malpe et al., 1997, and S. Mohan et al., 1991). In this context, the specific expression of the INSL4 gene during embryonic development in humans, both chronologically and in terms of its highly restrictive tissue distribution in the embryo, strongly suggests that this gene is very certainly involved in different stages of development of the forming bone and ligaments.

31

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Claims

34 CLAIMS

1. INSL4 gene, characterized in that it is expressed in fetal cells of bone tissue and / or ligaments.

2. EPIL 1, EPIL 2 or EPIL 3 polypeptide encoded by the INSL4 gene according to claim 1, characterized in that it is expressed in fetal cells of bone tissue.

3. Polypeptide according to claim 2, characterized in that the fetal cells of the bone tissue are cells of the perichondrium.

4. EPIL 1, EPIL 2 or EPIL 3 polypeptide encoded by the INSL4 gene according to claim 1, characterized in that it is expressed in fetal ligament cells.

5. Polypeptide according to one of claims 2 to 4, characterized in that it is involved in the process of differentiation of bone tissue cells and / or ligaments.

6. Polypeptide according to one of claims 2 to 4, characterized in that it is involved in the process of proliferation of bone tissue cells and / or ligaments.

7. Polypeptide according to one of claims 2 to 4, characterized in that it is involved in the process of regeneration of bone tissue cells and / or ligaments.

8. Polypeptide according to one of claims 2 to 7, characterized in that it is involved in the process of development of the cartilage of the bone in formation.

9. Polypeptide according to one of claims 2 to 7, characterized in that it is involved in the process of ossification of the bone in formation.

10. Use of one or more antibodies directed against an EPIL 1, EPIL 2, EPIL 3 polypeptide according to one of claims 2 to 9, or one of their homologs or variants, or against one of their fragments , for the detection, identification, localization and / or assay of an EPIL 1, EPIL 2, EPIL 3 polypeptide or one of their homologs or variants, in bone tissue and / or in ligaments. 35

11. Use of one or more antibodies directed against an EPIL 1, EPIL 2, EPIL 3 polypeptide according to one of claims 2 to 9, or one of their homologs or variants, or against one of their fragments for the diagnosis of pathologies linked to the abnormal expression of the EPIL 1, EPIL 2 and / or EPIL 3 polypeptide in bone tissue and / or in ligaments.

12. Use of an oligonucleotide probe or primer for the detection, identification, localization, amplification and / or assay of nucleic acid sequence corresponding to the INSL4 gene according to claim 1 in bone tissue and / or in the ligaments.

13. Use of an oligonucleotide probe or primer capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene according to claim 1, for the diagnosis of pathologies linked to the abnormal expression of the INSL4 gene in bone tissue and / or in the ligaments.

14. Method for the detection, identification, localization and / or specific assay of an EPIL 1, EPIL 2, EPIL 3 polypeptide according to one of claims 2 to 9, or one of their homologs or variants, in a biological sample of bone tissue and / or ligaments, or for the diagnosis of pathologies linked to the abnormal presence of said polypeptide (s) in bone tissue and / or in ligaments, characterized in that it comprises the following stages: a) bringing said biological sample into contact with one or more antibodies directed against said polypeptide (s) or against one of their fragments; b) detection, identification, location and / or determination of the antigen-antibody complex formed.

15. Kit or kit for the detection, identification, localization and / or specific assay of an EPIL 1, EPIL 2, EPIL 3 polypeptide according to one of claims 2 to 9, or one of their counterparts or variants, in a biological sample of bone tissue and / or ligaments, or for the diagnosis of pathologies linked to the abnormal presence of said polypeptide (s) in said biological sample, characterized in that it comprises the following elements: a) a or several antibodies directed against the said polypeptide or polypeptides, or a fragment thereof; 36

b) where appropriate, the reagents for constituting the medium suitable for the immunological reaction; c) where appropriate, the reagents for the detection, identification and / or determination of the antigen-antibody complexes produced by the immunological reaction.

16. A method of detection, identification and / or specific assay of nucleic acid sequence corresponding to the INSL4 gene according to claim 1, in a biological sample of bone tissue and / or ligaments, or of diagnosis of pathologies linked to the abnormal presence of said nucleic acid sequence in said biological sample, characterized in that it comprises the following steps: a) isolation of the DNA from said biological sample to be analyzed, or obtaining a cDNA from the RNA of said biological sample; b) specific amplification of said nucleic acid sequence using at least one primer capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene; c) qualitative and / or quantitative analysis of the amplification products.

17. A method of detection, identification and / or specific assay of nucleic acid sequence corresponding to the INSL4 gene according to claim 1, in a biological sample of bone tissue and / or ligaments, or of diagnosis of pathologies linked to the abnormal presence of said nucleic acid sequence in said biological sample, characterized in that it comprises the following stages: a) contacting an oligonucleotide probe capable of specifically hybridizing with a corresponding nucleic acid sequence to the INSL4 gene with said biological sample, the DNA contained in said biological sample having, where appropriate, previously made accessible to hybridization, under conditions allowing hybridization of said probe to the DNA contained in said biological sample ; b) detection, identification and / or assay of the hybrid formed between said oligonucleotide probe and the DNA of said biological sample.

18. A method of detection, identification and / or specific assay of nucleic acid sequence corresponding to the INSL4 gene according to claim 1, in a biological sample of bone tissue and / or ligaments, or of diagnosis of 37

pathologies linked to the abnormal presence of said nucleic acid sequence in said biological sample, characterized in that it comprises the following stages: a) contacting an oligonucleotide probe immobilized on a support and capable of hybridizing specifically with a nucleic acid sequence corresponding to the INSL4 gene, with said biological sample, the DNA of the sample, having, if necessary, been previously amplified using at least one primer capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene and / or made accessible to hybridization, under conditions allowing hybridization of said probe to the DNA of said biological sample; b) bringing the hybrid formed between said oligonucleotide probe immobilized on a support and the DNA contained in said biological sample into contact, if appropriate after removing the DNA from said biological sample which has not hybridized with said probe, with an oligonucleotide probe, in particular labeled, and capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSLA gene.

19. Kit or kit for the detection, identification and / or specific assay of nucleic acid sequence corresponding to the INSL4 gene according to claim 1, in a biological sample of bone tissue and / or ligaments, or for diagnosis pathologies linked to the abnormal presence of said nucleic acid sequence in said biological sample, characterized in that it comprises the following elements: a) an oligonucleotide probe capable of specifically hybridizing with a nucleic acid sequence corresponding to INSL4 gene; b) where appropriate, the reagents necessary for carrying out a hybridization reaction; c) where appropriate, at least one primer capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene as well as the reagents necessary for a DNA amplification reaction. 38

20. Kit or kit for the detection, identification and / or specific assay of nucleic acid sequence corresponding to the INSL4 gene according to claim 1, in a biological sample of bone tissue and / or ligaments, or for diagnosis pathologies linked to the abnormal presence of said nucleic acid sequence in said biological sample, characterized in that it comprises the following elements: a) an oligonucleotide probe, called capture probe, which can be supplied pre-immobilized on a support, and capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene; b) an oligonucleotide probe, called a revelation probe, in particular labeled, and capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene; c) where appropriate, at least one primer capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene as well as the reagents necessary for a DNA amplification reaction.

21. Method or kit according to one of claims 14 to 20, for the diagnosis of pathologies linked to the differentiation and / or the abnormal proliferation of bone tissue and / or ligament cells.

22. The method or kit as claimed in claim 21, for the diagnosis of pathologies linked to the abnormal development of cartilage and / or to an abnormal ossification of the bone in formation.

23. Method or kit according to one of claims 14 to 20, for the diagnosis of osteoporosis and / or dysplasia.

24. Method for labeling a bone tissue and / or ligament cell, characterized in that it comprises the following steps: a) bringing a biological sample of bone tissue and / or ligaments into contact with one or more antibodies directed against the polypeptide (s) EPIL 1, EPIL 2 and / or EPIL 3 according to one of claims 2 to 9, said antibodies being capable of being labeled; b) where appropriate, labeling of said antibodies using labeled compounds capable of recognizing said antibodies. 39

25. Method for labeling a bone tissue and / or ligament cell, characterized in that it comprises the following steps: a) bringing a biological sample of bone tissue and / or ligaments into contact with one or more probes oligonucleotides capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene according to claim 1, said probes being capable of being labeled; b) where appropriate, labeling of said probes using labeled compounds capable of recognizing said probes.

26. A cell labeling method according to one of claims 24 and 25, characterized in that the bone tissue and / or ligament cell is a fetal cell.

27. Method for detecting abnormal cells of bone tissue and / or ligaments, characterized in that it implements a method according to one of claims 24 to 26.

28. Method for selecting a chemical or biochemical compound capable of modulating the differentiation, regeneration and / or proliferation of human and / or animal cells of bone tissue and / or ligaments, characterized in that it uses a polypeptide EPIL 1, EPIL 2, EPIL 3 according to one of claims 2 to 9, one of their homologous or variant polypeptides, or one of their fragments, a nucleic acid sequence corresponding to the INSL4 gene, or a bone tissue cell or ligaments, especially fetal.

29. Selection method according to claim 28, characterized in that said selected compound is capable of inhibiting the differentiation, regeneration and / or proliferation of human and / or animal cells of bone tissue and / or ligaments.

30. Selection method according to claim 28, characterized in that said selected compound is capable of promoting the differentiation, regeneration and / or proliferation of human and / or animal cells of bone tissue and / or ligaments.

31. Selection method according to one of claims 28 to 30, characterized in that said cells are fetal cells. 40

32. Selection method according to claim 31, characterized in that the fetal cells are cells of the perichondrium.

33. Chemical or biochemical compound, characterized in that it is capable of being selected by a method according to one of claims 28 to 32.

34. A compound according to claim 33, as a medicament.

35. Compound according to one of claims 33 and 34, characterized in that it is chosen from: a) an EPIL 1, EPIL 2 or EPIL 3 polypeptide, one of their homologous or variant polypeptides or one of their fragments, which can be obtained by genetic recombination or by chemical synthesis; b) an antibody directed against a polypeptide as defined in a); c) a chemical or biochemical compound, characterized in that it is a ligand of a polypeptide as defined in a); d) a vector comprising all or part of a nucleic acid sequence corresponding to the INSL4 gene; e) a vector as defined in d), having on its surface a specific marker of bone tissue cell or target ligaments whose activity is sought to modulate; f) a sense or antisense nucleic acid sequence capable of hybridizing specifically with a nucleic acid sequence corresponding to the INSL4 gene.

36. Compound according to one of claims 34 to 35, intended for the treatment of pathologies linked to the differentiation and / or the abnormal proliferation of bone tissue and / or ligament cells.

37. Compound according to claim 36, intended for the treatment of pathologies linked to the abnormal development of cartilage and / or to an abnormal ossification of the bone in formation.

38. Compound according to one of claims 34 to 35, intended for the treatment of osteoporosis or dysplasia.

39. Compound according to one of claims 34 to 35, intended for the treatment of pathologies involving the regeneration of bone tissue and / or ligaments. 41

40. Compound according to claim 39, characterized in that said treatment aims to repair lesions of the bone tissue and / or of ligaments following a traumatic shock.