WO2009019254A1 - Igfbp-2 c-terminal fragments and uses thereof - Google Patents

Abstract

There is increasing evidence that insulin-like growth factor (IGF)/insulin-like growth factor binding proteins exert cell-type specific IGFindependent effects in addition to IGF-modulating activty. Here, carboxyterminal IGFBP-2 fragments, IGFBP-2167-279, IGFBP-2167-289 and IGFBP- 2104289, were shown to exert a strong mitogenic effect on growth plate chondrocytes. Thus, a novel role of IGFBP-2 fragments is to effectuate endocrine and paracrine/autocrine regulation of longitudinal growth.

Description

IGFBP-2 C-TERMINAL FRAGMENTS AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to carboxy-terminal fragments of insulin-like growth factor binding protein-2 and uses thereof, especially in the development of alternative methods for treating GH-resistant types of microsomia, such as dwarfism.

BACKGROUND OF THE INVENTION

The insulin-like growth factor (IGF)/insulin-like growth factor binding protein (IGFBP) system is an important component in the hormonal regulation of longitudinal growth. These proteins have individual and celltype specific expression patterns and exert different functions including stimulation or inhibition of IGF bioactivity as well as IGF- independent actions (1). In the postnatal period, IGFBP-2 is the second most abundant IGFBP in the circulation and is present in various biological fluids (2,3). In the intact growth plate of mice, exclusively IGFBP-2 mRNA, in conjunction with IGF-I and IGF-II mRNA was detectable in the proliferative and prehypertrophic zone by in situ hybridization, suggesting a functional role of IGFBP-2 for the regulation of longitudinal growth (4). In rat growth plate chondrocytes in primary culture, IGFBP-2 mRNA is expressed under baseline conditions, and IGFBP-2 protein is the second most abundant IGFBP in conditioned cell culture medium of chondrocytes (5). In in vitro studies, IGFBP-2 exerts inhibitory as well as stimulatory effects on cell proliferation, which are cell-type specific (2,3,6). For example in growth plate chondrocytes in primary culture, intact IGFBP-2 inhibits IGF-I-induced cell proliferation (6). On the other hand, increased expression of IGFBP-2 is associated with enhanced growth of tumor cells (1). Transgenic mice overexpressing IGFBP-2 displayed significantly reduced gain of body weight, suggesting that IGFBP-2 is a negative regulator of normal somatic growth (7). IGFBP-2 has been demonstrated to exert specific effects on bone, since IGFBP-2 transgenic mice have reduced bone size and mass, but not density (8).

Proteolysis of IGFBPs in the circulation and tissues is an essential mechanism to regulate the bioavailability and half-live of IGFs. The presence of active IGFBP-2 fragments with partial IGF-binding activity was first described in human milk (9). IGFBP-2 fragments, which exhibited molecular masses of 12.7 and 12.9 kDa and started with GIy 169 and GIy 167, were isolated by Stanndker et al from human hemoftrate (10). In consecutive studies, a peptide library generated from human hemofiltrate was immunologically screened and 18 different IGFBP-2 fragments were isolated and characterized (11). All tested fragments retained low IGF-binding capacity and exhibited the integrin-binding sequence. The most abundant fragment IGFBP-21⁶⁷-²⁷⁹ showed 10% of IGF-II binding compared to recombinant human IGFBP-2 (11).

Because of the potential importance of IGFBP-2 for the regulation of longitudinal growth, the aim of this study was to investigate the mitogenic effect of intact and fragmented forms of IGFBP-2 in two cell culture models of the growth plate, rat growth plate chondrocytes in primary culture and the mesenchymal RCJ3.1C5.18 (RCJ) cell line. RCJ cells, derived from fetal rat calvaria (12, 13), do not express

IGF-I; therefore the action of this hormone can be studied without interference from endogenous IGFs (14).

The biological activity of three defined carboxy-terminal fragments was analyzed, IGFBP-2^167"279, IGFBP-2^167"279 and IGFBP-2^104"289' which were isolated from human hemofiltrate (11), in comparison to intact IGFBP-2 and IGF-I. Furthermore, we sought to identify the mechanisms by which intact and fragmented IGFBP-2 exert their different biological effects on growth plate chondrocytes. We focused on the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAP/ERK) 1/2 and focal adhesion kinase (FAK) intracellular signaling pathways, because previous data in other cell culture models had shown that IGFBP-2 interacts withvarious integrin receptors, which signal preferably through the MAP/ERK 1 /2 and FAK pathways (15). SUMMARY OF THE INVENTION

One aspect of the present invention is a method for treating a disease, comprising administering to an individual a carboxy-terminal fragment of insulin-like growth factor binding protein-2 (IGFBP-2), wherein the stimulatory activity of the fragment causes proliferation of chondrocytes. In one embodiment, the fragment of IGFBP-2 is a fragment of SEQ ID NOs. 4 or 5. In one embodiment, the fragment is selected from the group consisting of residues 104-289 of IGFBP-2, residues 167-287 of IGFBP-2, and residues 167-289 of IGFBP-2. In a further embodiment, the sequence of residues 104289 of IGFBP-2 is depicted in SEQ ID NO. 1, the sequence of residues 167287 of IGFBP-2 is depicted in SEQ ID NO. 2, and the sequence of residues 167-289 of IGFBP-2 is depicted in SEQ ID NO. 3.

In one embodiment, the disease is selected from the group consisting of muscle wasting, osteoporosis, diabetes mellitus, amyotrophic lateral sclerosis, peripheral and central neuropathies, inflammatory processes, dysregulated inflammatory reactions, malignancies, inflammatory and neoplastic diseases, growth disorders, diseases of the musculatur, osteologic disorders, disturbances of wound healing and bone fracture healing, disorders of the nervous system, disorders of the lymph organs, disorders of the stomach, and cancer.

Another aspect of the present invention is a medicament, comprising (1) at least one carboxy-terminal fragment of IGFBP-2 and (2) a pharmaceutical carrier for facilitating delivery of the medicament by any one of oral, intravenous, intramuscular, intracutane, intrathekalen application or as aerosol routes of administration.

In one embodiment, the medicament comprises an IGFBP-2 fragment that is a carboxy-terminal fragment of either SEQ ID NOs. 4 or 5. In one embodiment, the carboxy-terminal fragment causes chondrocyte cells to proliferate. In another embodiment, the IGFBP-2 fragment comprises the sequence depicted in any one of SEQ ID NOs. 1-3, or any fragment thereof, wherein the stimulatory activity of the fragment causes proliferation of chondrocytes.

Another aspect of the present invention is a method for diagnosing end-stage renal disease in an individual, comprising determining whether an individual has an elevated level of at least one IGFBP-2 fragment in a serum sample taken from the individual, wherein an elevated level of the fragment in the serum compared to a serum sample from a control healthy individual indicates the individual has a form of end-stage renal disease.

In one embodiment, the serum level of at least one of SEQ ID NOs. 13 is determined and compared to a control healthy serum sample. In one embodiment, the individual is a mammal. In one embodiment, the individual is a human, dog, cat, cattle, pig, sheep, rat, mouse, guinea pig, hamster, horse, cow, chicken, rodent. In another embodiment, the individual is a human. In one embodiment, the human is a man, woman, child, unborn child, fetus, or embryo. In a particular embodiment, the human child is a pre-pubescent child.

Another aspect of the present invention is the use of an IGFBP-2 carboxy-terminal fragment for the preparation of a medicament for treating a disorder in an individual, wherein the disorder is selected from the group consisting of muscle loss and weakness, osteoporosis, diabetes, amyotrophic lateral sclerosis, peripheral and central neuropathies, inflammatory processes, disturbed inflammation reactions, tumor illnesses, inflammatory and neoplastic illnesses, growth disturbance, illness of the musculature, disorders of the bone apparatus, wound and bone healing, disorders of the nervous system, disorders of the lymph organs, disorders of the stomach, and cancer. In one embodiment, the fragment of IGFBP-2 is a fragment of SEQ ID NOs. 4 or 5. In one embodiment, the fragment is selected from the group consisting of residues 104-289 of IGFBP-2, residues 167-287 of IGFBP-2, and residues 167-289 of IGFBP-2. In a further embodiment, the sequence of residues 104-289 of IGFBP-2 is depicted in SEQ ID NO. 1, the sequence of residues 167-287 of IGFBP-2 is depicted in SEQ ID NO. 2, and the sequence of residues 167-289 of IGFBP-2 is depicted in SEQ ID NO. 3.

According to the present invention, any carboxy-terminal fragment of IGFBP-2 may be used to treat or diagnose a particular disorder in an individual. In one embodiment, this means a fragment of SEQ ID NOs. 4 or 5. In another embodiment, the sequence of the fragment is depicted in SEQ ID NOs. 1-3, or by a sequence that is similar to, or otherwise shares sequence identity with, the sequence of any one of SEQ ID NOs. 1-3. Similarity between two polypeptides is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide. An amino acid of one polypeptide is similar to the corresponding amino acid of a second polypeptide if it is identical or a conservative amino acid substitution. Conservative substitutions include those described in Dayhoff, M.O., ed ., The Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation, Washington, D. C. (1978), and in Argos, P. (1989) EMBO J. 8: 779-785. For example, amino acids belonging to one of the following groups represent conservative changes or substitutions:

- Ala, Pro, GIy, GIn, Asn, Ser, Thr:

- Cys, Ser, Tyr, Thr:

- VaI, He, Leu, Met, Ala, Phe; - Lys, Arg, His;

- Phe, Tyr, Trp, His; and

- Asp, GIu.

Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2 : 482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J. MoI . Biol . 48: 443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Natl . Acad . Sci. 85 : 2444 (1988); by computerized implementations of these algorithms, including, but not limited to : CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, California; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin, USA; the CLUSTAL program is well described by Higgins and Sharp, Gene 73 : 237-244 (1988); Higgins and Sharp, CABIOS 5 : 151-153 (1989); Corpet et al ., Nucleic Acids Research 16: 10881-90 (1988); Huang et al.,

Computer Applications in the Biosciences 8: 155-65 (1992), and Pearson et al ., Methods in Molecular Biology 24: 307331 (1994).

The BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; 'IBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); Altschul et al., J. Mot. Biol.,

215 :403-410 (1990); and, Altschul et al., Nucleic Acids Res. 25 : 3389-3402 (1997).

Software for performing BLAST analyses is publicly available, e.g ., through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased .

Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when : the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T. and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M = 5, N = -4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. ScL USA 89 : 10915).

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90 : 58735877 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.

Multiple alignment of the sequences can be performed using the CLUSTAL method of alignment (Higgins and Sharp (1989) CABIOS. 5 : 151-153) with the default parameters (GAP PENALTY= IO, GAP LENGTH PENALTY= IO) . Default parameters for pairwise alignments using the CLUSTAL method are KTUPLE 1, GAP PENALTY=3, WINDOW = 5 and DIAGONALS SAVED = 5.

The following running parameters are preferred for determination of alignments and similarities using BLASTN that contribute to the E values and percentage identity for polynucleotide sequences: Unix running command : blastall -p blastn -d embldb -e 10 -GO -EO -r 1 -v 30 -b 30 -i queryseq -o results; the parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -r Reward for a nucleotide match (blastn only) [Integer]; -v Number of one-line descriptions (V) [Integer]; -b Number of alignments to show (B) [Integer]; -i Query File [File In]; and -o BLAST report Output File [File Out] Optional.

The "hits" to one or more database sequences by a queried sequence produced by BLASTN, FASTA, BLASTP or a similar algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.

The BLASTN, FASTA and BLASTP algorithms also produce "Expect" values for alignments. The Expect value (E) indicates the number of hits one can "expect" to see over a certain number of contiguous sequences by chance when searching a database of a certain size. The Expect value is used as a significance threshold for determining whether the hit to a database, such as the preferred EMBL database, indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the EMBL database, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. By this criterion, the aligned and matched portions of the polynucleotide sequences then have a probability of 90% of being the same. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in the EMBL database is 1% or less using the BLASTN or FASTA algorithm. According to one embodiment, "variant" polynucleotides, with reference to each of the polynucleotides of the present invention, preferably comprise sequences having the same number or fewer nucleic acids than each of the polynucleotides of the present invention and producing an E value of 0.01 or less when compared to the polynucleotide of the present invention. That is, a variant polynucleotide is any sequence that has at least a 99% probability of being the same as the polynucleotide of the present invention, measured as having an E value of 0.01 or less using the BLASTN, FASTA, or BLASTP algorithms set at parameters described above. Accordingly, an IGFBP-2 fragment sequence of the present invention may share 60% sequence identity, at least about 70% sequence identity, at least about 80% sequence identity, at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or at least about 100% sequence identity to any one of the IGFBP-2 carboxy-terminal sequences, or to any one of SEg ID NOs. 1-3.

The length of a carboxy-terminal IGFBP-2 fragment of the present invention may be about 10 residues long, about 20 residues long, about 30 residues long, about 40 residues long, about 50 residues long, about 60 residues long, about 70 residues long, about 80 residues long, about 90 residues long, about 100 residues long, about 110 residues long, about 120 residues long, about 130 residues long, about 140 residues long, about 150 residues long, about 160 residues long, about 170 residues long, about 180 residues long, about 190 residues long, about 200 residues long, about 210 residues long, about 220 residues long, or longer.

Another aspect of the present invention is an isolated peptide fragment of IGFBP-2, comprising the amino acid sequence of SEQ ID NO. 1, or a sequence that shares at least about 60% sequence identity, at least about 70% sequence identity, at least about 80% sequence identity, at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or at least about 100% sequence identity with the full-length sequence of SEQ ID NO. 1.

Another aspect of the present invention is an isolated peptide fragment of IGFBP-2, comprising the amino acid sequence of SEQ ID NO. 2, or a sequence that shares at least about 60% sequence identity, at least about 70% sequence identity, at least about 80% sequence identity, at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or at least about 100% sequence identity sequence identity with the full-length sequence of SEQ ID NO. 2.

Another aspect of the present invention is an isolated peptide fragment of IGFBP-2, comprising the amino acid sequence of SEQ ID NO. 3, or a sequence that shares at least about 60% sequence identity, at least about 70% sequence identity, at least about 80% sequence identity, at least about 90% sequence identity, at least about

91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or at least about 100% sequence identity the full-length sequence of SEQ ID NO. 3. Another aspect of the present invention also encompasses an isolated peptide fragment of IGFBP-2. In one embodiment, the fragment comprising residues 140-289 of IGFBP-2. In one embodiment, the IGFBP-2 comprises the sequence of SEQ ID NOs. 4 or 5.

Another aspect of the present invention is an isolated peptide fragment of IGFBP-2, consisting essentially of the amino acid sequence of SEQ ID NO. 1, or a sequence that shares at least about 60% sequence identity, at least about 70% sequence identity, at least about 80% sequence identity, at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or at least about 100% sequence identity with the full-length sequence of SEQ ID NO. 1.

Another aspect of the present invention is an isolated peptide fragment of IGFBP-2, consisting essentially of the amino acid sequence of SEQ ID NO. 2, or a sequence that shares at least about 60% sequence identity, at least about 70% sequence identity, at least about 80% sequence identity, at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or at least about 100% sequence identity sequence identity with the full-length sequence of SEQ ID NO. 2.

Another aspect of the present invention is an isolated peptide fragment of IGFBP-2, consisting essentially of the amino acid sequence of SEQ ID NO. 3, or a sequence that shares at least about 60% sequence identity, at least about 70% sequence identity, at least about 80% sequence identity, at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or at least about 100% sequence identity the full-length sequence of SEQ ID NO. 3. Another aspect of the present invention also encompasses an isolated peptide fragment of IGFBP-2. In one embodiment, the fragment comprising residues 140-289 of IGFBP-2. In one embodiment, the IGFBP-2 comprises the sequence of SEQ ID NOs. 4 or 5.

Another form of variant of any of the IGFBP-2 peptide fragments disclosed herein is one that is chemically, biochemically, or biologically modified in some way. Hence, the present invention encompasses fragments of IGFBP-2 that are biologically active fragments, and/or variants or derivatized versions, for example amidated, acetylated, sulfated, phosphorylated and or glycosylated and peptides. Any of such peptides also can be coupled to polyethylene glycol . Methods for chemically conjugating, synthesizing, or modifying a peptide are well know to the person of skill in the art and are standard laboratory techniques.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. IGFBP-2 fragments are present in normal serum and elevated in end-stage renal disease (ESRD) serum. Western immunoblotting of intact and fragmented IGFBP-2 in serum of children with ESRD (lane 3; 4) and age-matched controls (lane 1; 2). Intact human IGFBP-2 served as control (C). Two pi serum were separated on 12 % SDS-PAGE and immunostained using a human anti-IGFBP-2 antibody. ST = molecular weight marker.

Figure 2. Defined carboxy-terminal IGFBP-2 fragments, but not intact IGFBP-2, stimulate proliferation of growth plate chondrocytes in primary culture. Effect of intact IGFBP-2 (A), IGFBP-2^104"289 (B), IGFBP-2^167"289 (C), and of IGFBP-2^167"279 (D) alone (7.8 nM) and in coincubation with equimolar concentrations of IGF-I on DNA synthesis, as assessed by [³H)thymidine incorporation assay. Subconfluent chondrocytes in primary culture were cultured in serum-free medium for 24 hours. Medium was changed to F12/DMEM containing 0.2% BSA, and peptides or vehicle were added as indicated. [³H)thymidine incorporation was determined as described in the Materials and Methods section . [³H)thymidine incorporation in control cultures was 22165 ±

1884 cpm (range, 7167 to 41225 cpm). Data are the mean ± SE expressed as percent of control. Statistics by ANOVA of 24 parallel dishes per group. Experiments were performed at least three times. *P < 0.05 vs. control, #P < 0.05 vs. IGF-I.

Figure 3. Defined carboxy-terminal IGFBP-2 fragments, but not intact IGFBP-2, stimulate proliferation of RCJ cells. Effect of intact IGFBP-2 (A), IGFBP-2^104"289 (B)_,

IGFBP-2^167"289 (C), and of IGFBP-2^167"279 (D) alone (7.8 nM) and in coincubation with equimolar concentrations of IGF-I on DNA synthesis, as assessed by [³H]thymidine incorporation assay. Subconfluent RCJ cells were cultured in serum-free medium for 24 hours. Medium was changed to MEM containing 0.2% BSA, and peptides or vehicle were added as indicated. [³H]thymidine incorporation was determined as described in the Materials and Methods section. [³H]thymidine incorporation in control cultures was 30294 ± 3946 cpm (range, 9689 to 64159 cpm). Data are the mean ± SE expressed as percent of control. Statistics by ANOVA of 24 parallel dishes per group. Experiments were performed at least three times. *P < 0.05 vs. control, #P < 0.05 vs. IGF-I.

Figure 4. Subcellular distribution of intact and fragmented IGFBP-2. A, The biochemical fractions were isolated as described in the Material and Method section. To verify cell compartment separation, cell lysates from RCJ cells were run in parallel and probed for the cytoplasmic fraction with an antibody against GAPDH, for the plasma membrane preparation with an antibody against Na-K-ATPase, and for the nucleare fraction with an antibody against CREB B, Western immunoblot analysis of the respective cell compartments incubated with vehicle, intact or fragmented IGFBP-2 for 2 hours. The respective cell lysates of RCJ cells were run in parallel and probed with a specific antibody against IGFBP-2, as described in the Material and Method section.

Figure 5. Partially reduced full-length IGFBP-2 stimulates cell proliferation to a comparable extent as equimolar IGF-I. RCJ cells were cultured until confluence, serum-starved for 12 h and stimulated with IGF-I (7.8 nM) and intact IGFBP-2 (7.8 nM) incubated with or without DTT for additional 48 h. Control cells were cultured without IGF-I in the absence or presence of DTT (5 mM for 30 min). [³H]-thymidine incorporation into the acid-extractable pool was determined by scintillation counting and used as a measure of DNA synthesis, as described in Materials and Methods. Data are mean ± SE. Statistics by ANOVA were for 12 parallel wells per group from three independent experiments. *P< 0.05 vs. control; #P< 0.05 vs. IGF-I.

Figure 6. Both fragmented and partially reduced full-length IGFBP-2 stimulate the MAP/ERK 1/2 and FAK signaling pathways. A, Cells were serum-starved for 12 h and incubated for 1 h with the respective IGFBP-2 peptides: control (lane 1), intact IGFBP-2 (lane 2), IGFBP-2^104"289 (lane 3), IGFBP-2^167"289 (lane 4), IGFBP-2^167"279 (lane 5), and partially reduced full-length IGFBP-2 (lane 6). Cell lysates were subjected to Western immunoblot analysis, and the respective membranes were probed with specific antibodies against p-ERK and total ERK. Representative autoradiography of a total of three independent experiments are shown. B, After 12 h of starvation, cells were incubated for 1 h with the respective IGFBP-2 peptides: control (lane 1), intact IGFBP-2 (lane 2), IGFBP-2^104"289 (lane 3), IGFBP-2^167"289 (lane 4), IGFBP-2^167"279 (lane 5), partially reduced full-length IGFBP-2 (lane 6), and the respective membranes were probed with specific antibodies against p-FAK and total FAK. Representative blots of two independent experiments are shown.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to carboxy-terminal fragments of insulin-like growth factor binding protein-2, especially in the use of those fragments to develop alternative methods for treating GH-resistant types of microsomia, such as dwarfism. An aspect of the present invention, therefore, demonstrates that carboxy-terminal

IGFBP-2 fragments exert IGF-Iindependent stimulatory activity on the proliferation of both rat growth plate chondrocytes in primary culture and of RCJ cells.

An aspect of the present invention is the surprising discovery that treatment with carboxy-terminal fragments of IGFBP-2 effectuates a similar therapeutic efficacy as IGF-I, but avoids complications and side-effects such as the induction of hypoglycemia, pseudotumor cerebri and organ enlargement (liver, spleen, kidneys, heart). These problems can be avoided by administering any of the IGFBP-2 carboxy- terminal fragments or homologs disclosed herein alone or in combination with one another. Furthermore, the suppressive effect of rhIGF-I on growth hormone secretion which can have detrimental consequences on growth-hormone-regulated metabolic processes is not present on treatment with carboxy-terminal fragments of IGFBP-2. Hence, it is anticipated that the therapeutic index of carboxy-terminal fragments of IGFBP-2 is much more favorable than that for rhIGF-I. Moreover, the production and use of carboxy-terminal IGFBP-2 fragments is simple and cost effective.

Here, then, it is also shown that IGFBP-2 fragments are present in human serum and, for the first time, that a marked elevation of IGFBP-2 fragments in serum of children with end-stage renal disease (ESRD). An example of a full-length IGFBP-2 protein is found in GenBank Accession No. P18065 (Binkert et al., 1989, EMBO J. 8

(9), 2497-2502 (1989)), and as depicted in SEQ ID NOs 4 and 5 below.

Previous studies had shown an elevation of immunoreactive IGFBP-2 in serum of children (23, 24) and adults (25) with chronic renal failure, but it remained unclear to which extent intact and fragmented IGFBP-2 contributed to elevated immunoreactive IGFBP-2. Here, it is shown that both intact and fragmented IGFBP-2 are elevated to a similar extent in ESRD serum.

By Western immunoblotting, two IGFBP-2 fragments with a molecular mass of approximately 23 and 15 kDa could be detected in human serum. These fragments most likely correspond to IGFBP-2^104"289 (SEQ ID NO. 1) and IGFBP-2^167"289 (SEQ ID NO. 2), which had been previously isolated from a human hemofiltrate bank (11).

The presence of increased IGFBP-2 fragments is most likely due to decreased renal filtration by the diseased kidneys, but additional increased protease activity towards IGFBP-2 may also be operative. Fragments of IGFBP-3 are also increased in ESRD serum, which accumulate due to reduced renal clearance, while the protease activity towards IGFBP-3 is not increased in uremic serum (26).

Here, it is shown that intact IGFBP-2 inhibits IGF-I-dependent proliferation of growth plate chondrocytes in primary culture, confirming previous data from our laboratory (7); we have now extended this observation to RCJ cells. The inhibitory activity of IGFBP-2 appears to be due to sequestration of IGFs, because it binds to the IGFs with a higher affinity than the type 1 IGF receptor (27). In the absence of exogenous and/or endogenous (in RCJ cells) IGF-I, intact IGFBP-2 neither stimulated nor inhibited chondrocyte proliferation. IGF-independent stimulatory effects of intact IGFBP-2 on cell proliferation have been observed in nontransformed rat osteoblasts (28) and in the adrenocortical tumour cell line Y-I (2), while in intestinal epithelial cells (IEC-6) intact IGFBP-2 inhibited cell proliferation also in the absence of IGF-I (29). Direct evidence exists from experiments in transgenic mice that overexpressing IGFBP-2 is associated with reduced postnatal body weight gain (7), diminished bone volumes, (8) and inhibition of GH-stimulated growth in giant GH transgenic mice (30).

In contrast to intact IGFBP-2, all three IGFBP-2 fragments investigated did not inhibit IGF-I-dependent chondrocyte proliferation. This observation is most likely due to the reduced binding affinity of these fragments towards IGF-I. By saturation binding assays using [¹²⁵I)IGF-I, Mark et al demonstrated a 150-fold reduced IGF-I binding capacity for IGFBP-2^167"279 (SEQ ID NO. 3) (K_d = 495 nM) compared to intact IGFBP-2 (K_d= 3.4 nM) (11); the binding affinities for the other two fragments were not investigated in this study.

The main finding of this study was that all tested carboxy-terminal IGFBP-2 fragments had significant IGF-I-independent stimulatory activity on proliferation of both rat growth plate chondrocytes in primary culture and of RCJ cells. These congruent data indicate that the effect of IGFBP-2 fragments on chondrocyte proliferation applies to growth plate chondrocytes in general. This is the first report on the biological activity of naturally occurring defined IGFBP-2 fragments in cell culture. According to our observations, binding of fragmented IGFBP-2 to the cell membrane, internalization and transport to the nucleus are involved in the mechanism of the mitogenic effect of IGFBP-2 fragments. All three IGFBP-2 fragments investigated contain a RGD (Arg-Glu-Asp) motif within the residues 265-

267 (31) and a heparin-binding domain (PKKLRP) present within residues 180185 (32). It has been demonstrated that IGFBP-2 binds to the glycosaminoglycans chondroitin-4- and -6-sulfate and to the proteoglycan aggrecan (20), which are both molecular components of the chondrocyte cell membrane.

We provide here the first evidence that both intact and carboxyterminal fragments of IGFBP-2 are internalized into chondrocytes and transported to the nucleus. Internalization and nuclear transport of IGFBP-2 were observed also in other cell culture systems. Translocation of IGFBP-2 from the cytosol into the nucleus has been observed in lung adenocarcinoma cells (33) and in lung alveolar epithelial cells (34). Hoeflich et al . detected a 20 kDa IGFBP-2 fragment in the nuclear fraction of several tissues of transgenic IGFBP-2 mice (35). The mechanism by which IGFBP-2 is translocated to the nucleus is unknown, because IGFBP-2 lacks the classical nuclear localization signal, by which other IGFBPs such as IGFBP-3 and IGFBP-5 are capable of entering into the nucleus (36).

We observed for the first time that partially reduced full-length IGFBP-2 has the same mitogenic activity in growth plate chondrocytes as equimolar concentrations of IGF-I. We hypothesize that partial reduction by DTT exposes binding sites of IGFBP-2, which are otherwise covered in the folded protein. It will be the subject of future studies, whether and under which conditions partial reduction of full-length IGFBP-2 is also operative in vivo.

We found that both fragmented and partially reduced full-length IGFBP-2 exert their mitogenic effects by stimulation of the MAP/ERK 1/2 and FAK signaling pathways. Other investigators observed that IGFBP-2 promotes de-adhesion and reduced proliferation of a breast cancer cell line and a Ewing sarcoma cell line, associated with dephosphorylation of FAK and the p42/44 MAP-kinases (22). An early event in integrin signaling is marked by alterations in the phosphorylation status of FAK, which can be triggered by RGD-containing peptides such as IGFBP-2. This pathway is involved in the regulation of cell migration, apoptosis and cell growth (37). Hence, although the biological activity of IGFBP-2 differs in various cell culture systems, similar intracellular signaling pathways appear to be involved.

In summary, the defined carboxy-terminal fragments IGFBP-2^167"279 (e.g ., SEQ ID

NO. 3), IGFBP-2^167"289 (e.g., SEQ ID NO. 2), and IGFBP-2^104"289 (e. g . , SEQ ID NO. 1) which naturally occur in human serum and which are elevated in ESRD serum, exert a comparable mitogenic effect on growth plate chondrocytes as equimolar concentrations of IGF-I. Mechanisms involved include binding to the cell membrane, cell internalization and transport to the nucleus. The mitogenic activity of these IGFBP-2 fragments was mediated through activation of the MAP/ERK 1 /2 and FAK signaling pathways. These data imply a novel role of naturally occurring IGFBP-2 fragments for the endocrine and paracrine/autocrine regulation of longitudinal growth.

EXAMPLES

Example 1 - Reagents

Recombinant human IGF-I was purchased from Bachem (Heidelberg, Germany). [³H]thymidine (25 Ci/mmol) and ECL reagents were obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). PBS, HEPES, penicillin-streptomycin,

Ham's F-12 and DMEM were obtained from Seromed Biochrom KG (Berlin, Germany). BSA was purchased from Sigma-Aldrich Chemicals (Deisenhofen, Germany). MEM was purchased from cc Pro (Neustadt, Germany), foetal calf serum from Paa Laboratories (Pasching, Austria). Clostridium collagenase (EC 3.4.24.3), Deoxyribonuclease (DNAse I (EC 3.1.21.1) and trypan blue were from Roche

Diagnostics GmbH (Mannheim, Germany). Dithiothreitol (DTT) was purchased from invitrogen life technologies (Karlsruhe, Germany). The antibodies directed against phosphorylated ERK1/2, ERK1/2, phosphorylated FAK, FAK, CREB and the horseradish peroxidase-conjugated (antirabbit and antimouse) antibodies were from Cell Signaling Technology (Frankfurt am Main, Germany). The Attorney Docket No.

077964-0112 antibodies against Na-K-ATPase and GAPDH were from Upstate Cell Signaling Solutions (Charlottesville, USA) and Everest Biotech (Oxfordshire, UK), respectively. Anti-rat IGFBP-2 antibody and intact human IGFBP-2 were obtained from GroPep (Adelaide, Australia).

IGFBP-2 fragments (20 kDa IGFBP-2^104"289, 14 kDa IGFBP-2^167"289, 12.8 kDa

IGFBP-2^167"279) were purified from hemofiltrate of patients with end-stage renal disease (ESRD) as follows: isolation of IGFBP-2 fragments was guided by immunoblot-screening in fractions of a peptide library established from 10,000 liters of hemofiltrate obtained from patients suffering from ESRD as described previously (10). Immediately after blood filtration using ultrafiiters with a specified cut-off of 20 kDa, the filtrate was routinely chilled to 4°C and adjusted to pH 3 to prevent bacterial growth and proteolysis. For the first separation step the ultrafiltrate was applied to a strong cation-exchange column (Fractogel TSK SP 650(M), Merck, Darmstadt,

Germany) and peptides were batchwise eluted by means of a pH gradient. Each derived pH pool eluate was further separated by reversed phase chromatography resulting in a total of 350 different peptide containing fractions, which were analyzed for the presence of naturally occurring IGFBP-2 fragments by Western blotting (10). IGFBP immunoreactive fractions were further purified to homogeneity by analytical cation-exchange and reversed phase chromatography and were analyzed by electrospray mass spectrometry and conventional sequence analysis, as described previously (10).

Example 2 - Cell cultures

Epiphyseal chondrocytes from 60- to 80-g Sprague-Dawley rats (Charles River,

Kieslegg, Germany) were isolated and cultured as described previously (16,17). This study was approved by the Institutional Animal Care and Use Committee (35- 9185.81/102/98). Pooled growth plates from 4-8 animals were digested with clostridial collagenase (0.12% wt/vol) and bacterial DNAse (0.02% wt/vol.) in F-12 medium. Viability, determined after isolation and at the end of each experiment by the trypan blue exclusion technique, always exceeded 90%. Dissociated cells were counted using a Neubauer chamber (Scheik, Hofheim, Germany).

Cells were cultured in monolayers in 96-well plates for proliferation (Nunc, Wiesbaden, Germany) as described previously (16,17). The F12/DMEM (1 : 1) medium contained a nominal calcium concentration of 1.2 mM and the medium was supplemented with 10 mM HEPES, 100 μg/ml streptomycin, and 10% FCS. In previous studies using the same culture system, we demonstrated that the majority of cells after the first passage expressed typical markers for proliferative growth plate chondrocytes. Peptide hormones were dissolved in PBS and added every day unless indicated otherwise.

RCJ3.1 C5.18 cells (kindly provided by Dr. Anna Spagnoli, Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee, USA) were grown at 37°C in humidified 5% CO₂ atmosphere in MEM (with Earle's salts) supplemented with 1 mM N-acetyl-L-glutamine, 10 mM Hepes, 100 U/ml penicillin-streptomycin, 2 mM sodium pyruvate, 15% heat-inactivated fetal bovine serum, 10-7 M dexamethasone and studied within 25 passages. Cell viability was tested with the MTT assay. Cells were cultured in monolayers in 96-well plates for proliferation (Nunc, Wiesbaden, Germany) as described previously (18).

Example 3 - (³H)thymidine assay

Incorporation of [³H)thymidine into DNA was determined in 96-well plate cultures as uptake of radioactivity in trichloroacetic acid-precipitable material as described previously (16). Prior to the experiment, cells were synchronized in cell cycle by starving in serum-free F-12/DMEM for 24 h. Medium was changed to F-12/DMEM with 0.2% BSA, and hormones or solvents were added as indicated for 48 h. For the last 4 h, cultures were coincubated with 2 μCi of [³H[thymidine. Subsequently, cells were rinsed twice with phosphate-buffered saline and extracted with sodium hydroxide (1 M). Before counting the extract was mixed with scintillation fluid.

Example 4 - Western immunoblotting

RRCJ3.1 C5.18 cells were incubated with the indicated IGFBPs, scraped in 50 pi ice- cold lysis buffer containing a cocktail of proteinase and phosphatase inhibitors and cell extracts were treated as reported previously (19). The membranes were blocked either in 5% BSA or in 3% milk for 1 h at room temperature, incubated overnight with the first antibody (dilution 1 : 2000 in 5% BSA for p-ERKl/2, ERK1/2, FAK and p- FAK) washed extensively over a period of 30 min with Tris Buffer Saline-Tween 20

(TBS-T) 0.05%, and then incubated for 1 h with the secondary antibody (dilution 1 : 2000 in 3% milk), followed by further washing over a period of 30 min. The protein bands were visualized using a chemiluminescent detection system and Hyperfilm ECL film (Amersham Pharmacia Biotech, UK) according to the manufacturer's directions.

Example 5 - Membrane preparation

Membranes were prepared as described before (19). Tissues were homogenized in a lysis buffer (10 mM T ris, 2 mM phenylmethysulfonyl fluoride, 1 TIU/ml of aprotinin) for 1 min at 10 000 rpm on ice using a cell homogenizer (Micro-

Ultrasonic Cell Disrupter (Kimble/Kontes, Vineland, USA)). Cell debris and nuclei were separated by centrifugation (1000 x g. 10 min at 4° C). The supernatant containing plasma membranes and the cytosolic fraction were separated by centrifugation for 30 min at 21 000 x g at 4° C. The isolated plasma membranes were stained with the antibody for Na-K-ATPase (blocked with 3% milk and a dilution of 1 : 5000) and incubated for thirty minutes. The cytosolic fraction was stained with the antibody against GAPDH (blocked with 5% BSA and a dilution of 1 : 5000) and incubated for five minutes. The nuclear fraction was incubated with the antibody against CREB (blocked with 5% BSA and a dilution of 1 : 2000) and incubated for one hour.

Example 6 - Patients

Serum was obtained under outpatient conditions in the morning after an overnight fast. The children were studied after informed parental consent, and the study protocol was approved by the local Ethics Committee. The patients with ESRD were all on hemodialysis and compared with agematched controls. These patients were not suffering from additional thyroid, liver, or gastrointestinal disease, systemic diseases like lupus erythematosus, amyloidosis or oxalosis, severe cardiac diseases or treatment with glucocorticoids or other immunosuppressive drugs during the previous 6 months were excluded form the study. Patients with ESRD received medications consisting of vitamin D or cholecalciferol, water-soluble vitamins, oral phosphate binders, oral sodium bicarbonate, and no growth hormone (GH) therapy. Example 7 - Statistics

Data are given as mean ± SE. All data were examined for normal and non-Gaussian distribution by the Kolmogorov-Smirnov test. For comparison among normally distributed groups, one-way ANOVA followed by pairwise multiple comparison (Student-Newman-Keuls method) was used. For nonnormally distributed data, the non-parametric Kruskal-Wallis test followed by an all pairwise multiple comparison (Dunnetf's method) was used. P<0.05 was considered statistically significant.

Example 8 - IGFBP-2 fragments are present in normal serum and elevated in end-stage renal disease serum

In order to verify that low molecular weight fragments of IGFBP-2 are present in the circulation, Western immunoblotting of serum of children with ESRD and age- matched healthy controls was performed. A representative blot is shown in Figure 1. Both intact and fragmented IGFBP-2 were present in serum of normal children and markedly (up to five-fold) elevated in serum of children with ESRD. The detected IGFBP-2 fragments had molecular masses of approximately 15 kDa, consistent with the molecular mass of IGFBP-2^167"289 and 23 kDa, consistent with the molecular mass of IGFBP-2^104"289-

Example 9 - Defined carboxy-terminal IGFBP-2 fragments, but not intact IGFBP-2, stimulate growth plate chondrocyte proliferation

IGF-I in a concentration of 60 ng/ml (7.8 nM) stimulated proliferation of growth plate chondrocytes in primary culture by 1.8- to 2.5-fold (Fig. 2). We have shown previously that this IGF-I concentration maximally stimulates cell proliferation in this cell culture model (16). Intact IGFBP-2 had no effect on cell proliferation, while simultaneous exposure of cells to IGF-I and intact IGFBP-2 in equimolar concentrations reduced IGF-Iinduced cell proliferation by 50% (Fig . 2 A). In contrast to intact IGFBP-2, the carboxy-terminal fragment IGFBP-2^104"289 stimulated cell proliferation three-fold in the absence of exogenous IGF-I, but did not inhibit IGF-I- mediated cell proliferation (Fig . 2B). The effects of IGFBP-2^104"289 and IGF-I on cell proliferation were not additive, indicating that both peptides use similar intracellular signaling pathways. The IGFBP-2 fragments IGFBP-2^167"289 and IGFBP-2^167"279 also stimulated chondrocyte proliferation in the absence of exogenous IGF-I, although to a somewhat lesser degree, and did not modify IGF-I-induced cell proliferation (Fig . C and D).

To determine whether intact and fragmented IGFBP-2 exerts comparable effects in other cell culture models of the growth plate, similar experiments were performed in the mesenchymal chondrogenic cell line RCJ, derived from fetal rat calvaria (12, 13). In contrast to growth plate chondrocytes in primary culture, this cell line does not express IGF-I; therefore, the action of this hormone can be studied without interference from endogenous IGF-I (14). The results obtained with this cell culture model (Fig . 3) were well comparable to those obtained in growth plate chondrocytes in primary culture. We therefore performed the following experiments exclusively in RCJ cells.

Example 10 - Subcellular distribution of intact and fragmented IGFBP-2

Next we sought to analyze, whether this effect of IGFBP-2 fragments on cell proliferation is mediated by binding to the cell membrane and/or by cellular internalization. The separation of cells into defined compartments was verified by Western immunoblotting with antibodies against GAPDH as a specific marker for the cytosol, against Na-K-ATPase as a specific marker for the cell membrane and against

CREB as a specific marker for the nucleus. Na-K-ATPase was detected exclusively in the cell membrane fraction, CREB in the nuclear fraction and GAPDH in the cytosolic fraction (Fig 4 A). After incubation of cells with intact and fragmented IGFBP-2 over 2 hours, the localization of these peptides in the cell membrane, the cytoplasm and the nucleus was investigated by Western immunoblotting of different cell compartments with an antibody against rat IGFBP-2 (Fig. 4). Intact IGFBP-2 (32 kDa) was detected both in the plasma membrane and nuclear preparations, but not in the cytosolic fraction (Fig . 4 B) . A comparable pattern was obtained for the fragments IGFBP-2^104"289, IGFBP-2^167"289, and IGFBP-2^167"279. In control cells, the faint 32 kDa band most likely represents endogenous IGFBP-2. These findings demonstrate that intact IGFBP-2 and carboxy-terminal IGFBP-2 fragments are capable of binding to the cell membrane, are imported across the plasma membrane and transported to the nucleus.

Example 11 - Partially reduced full-length IGFBP-2 stimulates cell proliferation to a comparable extent as equimolar IGF-I

Accordingly, it is possible that although intact IGFBP-2 is localized in the same cellular compartments as fragmented IGFBP-2, it does not exert mitogenic activity, because its intact tertiary structure prevents it from binding to down-stream signaling molecules. To test this, intact IGFBP-2 was incubated with DTI", which destroys disulphide bonds of peptide hormones (21). As shown in Figure 5, partially reduced full-length IGFBP-2 stimulated cell proliferation to a comparable extent as equimolar IGF-I, while intact full-length IGFBP-2 or the addition of DTI' to control cells was ineffective.

In order to identify potential intracellular signaling pathways for the mitogenic effect of partially reduced full-length IGFBP-2, the MAP/ERK 1 /2 pathway was inhibited with the specific pharmacological inhibitor U0126 in an additional series of experiments. We focused on the MAP/ERK 1/2 pathway, because previous data in other cell culture models had shown that IGFBP-2 interacts with various integrin receptors, which signal preferably through the MAP/ERK 1 /2 pathway (22). Co- incubation of cells with U0126 completely abrogated the mitogenic effect of partially reduced full-length IGFBP-2 (Table 1), indicating that the MAP/ERK 1/2 signaling pathway is involved.

Table 1. Partially reduced full-length IGFBP-2 exerts its mitogenic effect through the MAP/ERK 1/2 pathway. Subconfluent chondrocytes were cultured in serum-free medium for 24 h. Medium was changed to F12/DMEM containing 0.2% BSA, and peptides or vehicle were added for 48 h as indicated. The sample with peptide and DTT was incubated with 5 mM DTT for 30 min. [³H]thymidine incorporation was determined as described in Materials and Methods. [³H]thymidine incorporation in control cultures was 342 ± 27.4 cpm (range, 216 - 450 cpm). Data are the mean ± SE expressed as the percent of control. Statistics by ANOVA of 9 to 12 parallel dishes per group. *P<0.05 vs. control; *P<0.05 vs. full-length IGFBP-2 + DTT.

Control + DTT (%) IGF-I (%) Intact IGFBP-2 Full-length IGFBP-2 Fulllength IGFBP-2

(%) + DTI¹ (%) + DTT + U0126 (%)

Control

(%)

100 ± 26 99 ± 12 367 ± 60* 124 ± 24 345 ± 38* 106 ± 26#

Example 12 - Carboxy-terminal IGFBP-2 fragments and partially reduced full- length IGFBP-2 stimulate the MAP/ERK 1/2 and FAK signaling pathways

Next, we sought to investigate whether IGFBP-2 activates key signaling molecules of the MAP/ERK 1/2 and FAK signaling pathways. Similarly as the MAP/ERK 1/2 pathway, the FAK pathway is activated by signaling through various integrin receptors (13). We observed that the three IGFBP-2 fragments IGFBP-2^104"289, IGFBP-2^167"289, and IGFBP-2^167"279 and also partially reduced full-length IGFBP-2 were capable of stimulating phosphorylation of ERIC, while intact IGFBP-2 was ineffective (Fig. 6 A). Similarly, the three IGFBP-2 fragments IGFBP-2^104"289, IGFBP-2^167"289, and IGFBP-2^167"279 and partially reduced full-length IGFBP-2, but not intact IGFBP-2, stimulated phosphorylation of FAK (Fig. 6 B). Taken together, these results indicate that the MAP/ERK 1/2 and FAK signaling pathways are involved in the mitogenic effect of carboxy-terminal IGFBP-2 fragments.

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SEQ ID NO. 1 (IGFBP-4, RESIDUES 104-289 OF IGFBP-2 , MW 20684.5)

104 - gaspeqva dngddhsegg lvenhvdstm nmlggggsag rkplksgmke lavfrekvte ghrgmgkggk hhlgleepkk lrpppartpc qqeldqvler istmrlpder gplehlyslh ipncdkhgly nikgckmsln ggrgecwcvn pntgkliqga ptirgdpech lfyneggear gvhtgrmq - 289

SEQ ID NO. 2 (IGFBP-4, RESIDUES 167-289 OF IGFBP-2, MW 14023.1)

167 - gkggk hhlgleepkk lrpppartpc qqeldqvler istmrlpder gplehlyslh ipncdkhgly nikgckmsln gqrgecwcvn pntgkliqga ptirgdpech lfyneggear gvhtgrmq - 289

SEQ ID NO. 3 (IGFBP-4, RESIDUES 167-279 OF IGFBP-2, MW 12 857.7)

167 - gkggk hhlgleepkk lrpppartpc qqeldqvler istmrlpder gplehlyslh ipncdkhgly nikgckmsln gqrgecwcvn pntgkliqga ptirgdpech lfynegge - 279

SEQ ID NO. 4 (IGFBP-2 HUMAN PRECURSOR, RESIDUES 1-39 SIGNAL PEPTIDE, RESIDUES 40-328 MATURE CHAIN)

1 - mlprvgcpal plppppllpl sgggggarae vlfrcppctp erlaacgppp 61 vappaavaav aggarmpcae lvrepgcgcc svcarlegea cgvytprcgq glrcyphpgs 121 elplgalvmg egtcekrrda eygaspeqva dngddhsegg lvenhvdstm nmlggggsag 181 rkplksgmke lavfrekvte ghrgmgkggk hhlgleepkk lrpppartpc qqeldqvler 241 istmrlpder gplehlyslh ipncdkhgly nikgckmsln gqrgecwcvn pntgkliqga 301 ptirgdpech lfyneggear gvhtgrmq - 328

SEQ ID NO. 4 (IGFBP-2 HUMAN MATURE CHAIN) 1 - e vlfrcppctp erlaacgppp vappaavaav aggarmpcae lvrepgcgcc svcarlegea cgvytprcgq glrcyphpgs elplgalvmg egtcekrrda eygaspeqva dngddhsegg lvenhvdstm nmlggggsag rkplksgmke lavfrekvte ghrgm gkggk hhlgleepkk lrpppartpc qqeldqvler istmrlpder gplehlyslh ipncdkhgly nikgckmsln gqrgecwcvn pntgkliqga ptirgdpech lfyneggear gvhtgrmq - 289

Claims

Claims

1. A method for treating a disease, comprising administering to an individual a carboxy-terminal fragment of insulin-like growth factor binding protein-2 (IGFBP-2), wherein the stimulatory activity of the fragment causes proliferation of chondrocytes.

2. The method of claim 1, wherein the fragment is selected from the group consisting of residues 104-289 of IGFBP-2, residues 167-287 of IGFBP-2, and residues 167-289 of IGFBP-2. 3. The method of claim 1 or 2, wherein the sequence of residues 104289 of

IGFBP-2 is depicted in SEQ ID NO. 1, the sequence of residues 167287 of IGFBP-2 is depicted in SEQ ID NO. 2, and the sequence of residues 167-289 of IGFBP-2 is depicted in SEQ ID NO.

3.

4. The method of any one of the claims 1 to 3, wherein IGFBP-2 comprises the amino acid sequence depicted in SEQ ID NO. 4.

5. The method of any one of the claims 1 to 4, wherein the disease is selected from the group consisting of muscle wasting, osteoporosis, diabetes mellitus, amyotrophic lateral sclerosis, peripheral and central neuropathies, inflammatory processes, dysregulated inflammatory reactions, malignancies, inflammatory and neoplastic diseases, growth disorders, diseases of the musculatur, osteologic disorders, disturbances of wound healing and bone fracture healing, disorders of the nervous system, disorders of the lymph organs, disorders of the stomach, and cancer.

6. The method of any one of the claims 1 to 5, wherein the carboxy-terminal fragment of insulin-like growth factor binding protein-2 is a biologically active fragment, or variant thereof.

7. The method of claim 6, wherein the variant is an amidated, acetylated, sulfated, phosphorylated, glycosylated, or polyethylene-coupled form of the carboxy- terminal fragment.

8. The method of any one of the claims 1 to 7, wherein the carboxy-terminal fragment of insulin-like growth factor binding protein-2 is a biologically active fragment, or variant thereof.

9. The method of claim 8, wherein the variant is an amidated, acetylated, sulfated, phosphorylated, glycosylated, or polyethylene-coupled form of the carboxy- terminal fragment.

10. A medicament, comprising (1) at least one carboxy-terminal fragment of IGFBP-2 and (2) a pharmaceutical carrier for facilitating delivery of the medicament by any one of oral, intravenous, intramuscular, intracutaneous, intrathekal application or as aerosol routes of administration.

11. The medicament of claim 10, wherein the IGFBP-2 fragment is a carboxy- terminal fragment of either SEQ ID NOs. 4 or 5, wherein the fragment causes chondrocyte cells to proliferate.

12. The medicament of claim 10 and/or 11, wherein the IGFBP-2 fragment comprises the sequence depicted in any one of SEQ ID NOs. 1-3, or any fragment thereof, wherein the stimulatory activity of the fragment causes proliferation of chondrocytes.

13. A method for diagnosing end-stage renal disease in an individual, comprising determining whether an individual has an elevated level of at least one IGFBP-2 fragment in a serum sample taken from the individual, wherein an elevated level of the fragment in the serum compared to a serum sample from a control healthy individual indicates the individual has a form of end-stage renal disease.

14. The method of claim 13, wherein the serum level of at least one of SEQ ID NOs. 1-3 is determined and compared to a control healthy serum sample.

15. The method of claim 13 and/or 14, wherein the individual is a human.

16. The method of claim 15, wherein the human is a human child.

17. The method of claim 16, wherein the child is a pre-pubescent child.

18. The use of an IGFBP-2 carboxy-terminal fragment for the preparation of a medicament for treating a disorder in an individual, wherein the disorder is selected from the group consisting of muscle wasting, osteoporosis, diabetes mellitus, amyotrophic lateral sclerosis, peripheral and central neuropathies, inflammatory processes, dysregulated inflammatory reactions, malignancies, inflammatory and neoplastic diseases, growth disorders, diseases of the musculatur, osteologic disorders, disturbances of wound healing and bone fracture healing, disorders of the nervous system, disorders of the lymph organs, disorders of the stomach, and cancer.

19. The use of claim 18, wherein the IGFBP-2 fragment is a carboxy-terminal fragment of either SEQ ID NOs. 4 or 5, wherein the fragment causes chondrocyte cells to proliferate.

20. The use of claim 19, wherein the IGFBP-2 fragment comprises the sequence depicted in any one of SEQ ID NOs. 1-3, or any fragment thereof.

21. An isolated peptide fragment of IGFBP-2, comprising the amino acid sequence of

SEQ ID NO. 1, or a sequence that shares at least about 90% sequence identity with the full-length sequence of SEQ ID NO. 1.

22. An isolated peptide fragment of IGFBP-2, comprising residues 140-289 of

IGFBP-2.

23. The isolated peptide fragment of claim 22, wherein the IGFBP-2 comprises the sequence of SEQ ID Nos. 4 or 5.