Growth Factors Zvi Nevo, PhD, Zvi Laron, MD

process of growth is still surrounded by an aura of mystery. The efforts of biologists the world over in their search to solve the puzzle of growth have provided us with blocks of knowledge to be placed here and there within a framework, but the picture is far from complete. The important role played by the pituitary-secreted growth hormone as well as by the hormones secreted by the thyroid, gonads, and pancreas has long been recognized. To these have now been added recent discoveries of other growth-promoting substances, which suggest a wide field for investigation by both biologists and clinicians.

The complex

Hormones, strictly speaking,

are

substances that act at a distance from the site of their synthesis. Since some of these recently discovered growth\x=req-\ promoting substances do not always comply with this criterion, many investigators refer to them as growth factors, although many use the term "factors" interchangeably with "hormones." For the purpose of this review, a growth factor will be considered to be any substance that acts (1) to increase cell size and rate of prolif¬ eration as well as matrix production, and (2) to prolong cell survival. The recent literature contains a number of reviews on the subject of growth factors14 but the rapid ad¬ vances being made in this new field mean that much of this material is already outdated. This report at¬ tempts to summarize for both the clinician and biologist the existing body of knowledge on the various positive and negative growth-regulat¬ ing substances, with the awareness From the Department of Chemical Pathology, Sackler School of Medicine, Tel Aviv University, Tel Hashomer (Dr Nevo), and the Institute of Pediatric and Adolescent Endocrinology, Beilinson Medical Center, Petah Tikva, Israel (Dr

Laron).

Reprint requests to Institute of Pediatric and Adolescent Endocrinology, Beilinson Medical Center, Petah Tikva, Israel (Dr Laron).

that by the time it goes to press it too may well be incomplete, another few bits having already been added to the puzzle by the literature. SOMATOMEDINS The somatomedins

(SMs)

are

a

family of polypeptides that have been

found to exhibit growth-promoting activity both in vivo and in vitro. The existence of these substances was discovered incidentally during the search for an improved bioassay for growth hormone.5 It was found that serum from normal rats stimulated sulfate incorporation into proteoglycans in the cartilage of hypophysectomized rats, whereas that from hypophysectomized rats failed to do so even when growth hormone was added to the incubation mixture. (Proteoglycans are macromolecules com¬ posed of moieties of protein less than 10% and carbohydrates more than 90% covalently bound. They are major constituents of the extracellular ma¬ trix of connective tissues.) This growth agent, first called a "sulfation factor," was later renamed somatomedin," a term denoting the relation to somatotrophin, or growth hormone (GH), and the intermediary role played by SM in transmitting the message of this hormone to the target cells. The first method for measuring SM was a bioassay based on the previously described method of using costal carti¬ lage from hypophysectomized rats. Washed slices of cartilage were incu¬ bated with labeled sulfate, with and without the serum to be tested. The SM in whole serum is expressed in units of activity per volume of tested plasma (1 mL), where one unit of activity represents the increase in radioactive sulfate ion (:15S04) incorpo¬ ration into cartilage proteoglycans in the presence of 1 mL of a reference serum obtained from a pool of normal human adult serum. Somatomedin in purified preparations is expressed as

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units of

activity per milligram of protein comparable to the activity in whole serum.7 15S04 incorporation is the combined result of a stimulatory effect by the SMs and an inhibitory effect by various substances both known (eg, corticosteroids) and un¬ known/ Thus, this assay measures the net SM activity but not the concentra¬ tion of SM. Modifications of the original bioas¬ say using cartilage from hypophysectomized rats have been proposed by various investigators. Hall" intro¬ duced a bioassay using cartilaginous pelvic leaflets from 11-day-old chick embryos; this is a relatively simple and cheap procedure and has approxi¬ mately the same precision as the rat bioassay. Assuming that cartilages from various sources respond to the

serum growth stimulants, a variety of animal cartilages have been given a trial, including those from the monkey, the horse, the calf, the dog, the pig, the sheep, and the rat, with no one species being observed to have a particular advantage over any of the same

others.1" Van den Brande and Du Cajú1' suggested the use of a porcine rib cartilage for a routine bioassay in view of the ready availability of ho¬ mologous cartilage slices. Another method studied the incorporation of labeled thymidine into cartilage and is based on the pleiotypic effect (the

ability

to

generate simultaneously

several different types of cellular activities) of SMs.12 Since all the described bioassays still leave much to be desired with respect to reproducibility and credability, efforts are continuously being made to achieve an improved assay. Recent investiga¬ tions have attempted to use a variety of isolated cell types, including fetal chondrocytes,13 fetal lung fibroblasts,11 and human glial cells,15 to measure SM activity by following the incorporation of labeled sulfate and thymidine into glycosaminoglycan

(macromolecules containing repeating

disaccharide units made of an N-Achexosamine and a uronic acid—a name replacing the old term "mucopolysaccharides") and DNA, respectively. The effectiveness of serum on such a variety of cells and the wide spec¬ trum of reactions initiated within the same cell raised the suspicion that there is more than one SM. Indeed, a series of low molecular weight peptides with growth-promoting activity were purified and characterized from Cohn's fraction IVb (blood plasma proteins and some globulins resulting from ethanol-low temperature-pHsalt concentration fractionation of whole plasma according to Cohn's method) obtained from the plasma of subjects without growth problems or acromegalie subjects1617 by various

procedures, including gel filtration, ion exchange resin chromatography, high-voltage electrophoresis, affinity chromatography, electrofocusing, and polycrylamide gel (a polymerized gel commonly used for separation of macromolecules) electrophoresis.

Three main types of SMs were identi¬ fied, SM-A, SM-B, and SM-C, as well as a number of other substances that may eventually be classified as SMs, and which will be discussed later. It is possible that there exists only one active SM, the other substances being precursors or metabolites. The availability of purified SMs made it possible to develop specific radioimmunoassays and radioreceptor assays,181" by which the distribution of SMs and the mechanism of their activity could be investigated. It was found that SMs are widely distrib¬ uted, being present in cartilage, mus¬ cle, the liver, the kidneys, the brain, the pancreas, the heart, and the pitui¬ tary gland, as well as in body fluids, including lymph, amniotic fluid, and urine.2""26 The liver and the kidneys were found to be the two major sites of control of serum SMs, the former being the center of synthesis and the latter being the main site of catabo¬ lismo7-28 In the liver, SMs were found free of carrier proteins, but in the blood¬ stream, they are associated with specific high molecular weight carrier proteins. The synthesis of both SM and its carrier proteins has been shown to be GH dependent.2" The

complex of SM-carrier protein is biologically inactive. The exact physi¬ ological conditions under which this complex dissociates to release the active low molecular weight SM are as yet unknown; it has been suggested that GH plays a role in the associa¬ tion-dissociation of the complex.3" The biological half-lives of both SM and its carrier protein have been shown to be

about 12 hours.31 Somatomedin has been reported to show both daily fluc¬ tuations and a diurnal rhythm with a noon peak and nocturnal low. These cycles have no obvious connection with the human growth hormone (HGH) secretion, which peaks at night and fluctuates after meals or exercise.,2 The SMs are believed to initiate activity by attaching themselves to specific receptor sites on the plasma membrane of cells", which sets off a series of reactions that lead to the so-called positive pleiotypic response (an ability to generate several differ¬ ent types of anabolic-synthetic pro¬ cesses). The degree of responsiveness of the cells depends on the number of receptor sites and their sensitivity, which vary with age, state of health and nutrition, and on the extent of regulation of the cells by other hormones.'1 The information available indicates that SM competes with insulin at its receptor sites.15 It is of interest that recent reports have suggested the existence of intracellular binding sites for SM similar to those described for steroids.1" Somatomedin-A

Somatomedin-A is a neutral peptide with an isoelectric point at a pH of 7.1 to 7.5 and a molecular weight of 7,600 daltons. On performance of gel elec¬ trophoresis, two separate bands have been found, SM-A, and SM-A2, that are equally active in the bioassay using embryonal chick pelvic rudi¬ ments.'637 The levels of SM-A (in serum) have been found to correspond with those of its protein carrier. Although the most common method for determining SM-A activity is the chick cartilage bioassay, a recently developed radioreceptor assay using human placental membranes'" is pref¬ erable in situations where inhibitors of SM-A are suspected. The avallabili-

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ty of purified SM-A and of its antiserum, however, is still limited to only a very few laboratories.

Somatomedin-A exerts an insulin¬ like activity in the adipose tissue and the isolated fat cells of rats, stimulat¬ ing glucose oxidation to CO,, sup¬ pressing glycerol release, and decreas¬ ing calcium uptake.38 Like the classic anabolic hormones, SM-A antagonizes the action of catabolic adenylatecyclase-activating hormones such as epinephrine in vitro.3" The measure¬ ment of serum SM-A, and possibly of SM-C (to be discussed later), consti¬ tutes one of the potentially most useful values for the assessment of

growth activity.

Considerable data have been col¬ lected on serum SM-A levels in man. As yet, there is only scanty informa¬ tion on the levels present in fetal life and the neonatal period'" and on their possible importance, but it has been found that adult basal levels, which range from 0.8 to 1.2 U/mL, are reached at about 4 to 8 years of age. There are contradictory reports on the degree of correlation between serum SM levels and sex, age, height, weight, growth, skeletal maturation, puberty, and aging." There is general agree¬ ment that SM-A levels decrease to less than 0.5 U/mL after performance of a hypophysectomy and in patients with liver disease or pituitary insufficien¬ cy."14 Other states in which the SM-A level has been found to be decreased include chronic malnutrition, particu¬ larly when due to a lack of protein (a state usually associated with high levels of plasma GH),45 anorexia nervosa, and psychosocial dwarfism.'6 Somatomedin-A levels also decrease when there is increased corticosteroid production, as in Cushing's syndrome, or after the administration of exoge¬ Of particular nous corticosteroids.47 interest among the conditions in which plasma SM-A levels are low is Laron-type dwarfism (a syndrome characterized by clinical resemblance to isolated GH deficiency but display¬ ing a high concentration of immunoreactive plasma GH and lack of SM). These children, who in clinical and laboratory aspects resemble children with pituitary dwarfism due to iso¬ lated HGH deficiency, have very low levels of SM-A but high plasma '8

concentrations of HGH.4" · ""' It is thought that the primary defect may reside in the liver, which may be incapable of synthesizing SM-A,'1 or in the HGH molecule, which may be biologically inactive.52 A few such chil¬ dren with low levels of serum SM-A but with normal levels of HGH whose conditions did respond to exogenous HGH, are suspected to have a biologi¬ cally inactive HGH with no defect in SM-A generation.53 In states of GH deficiency, the administration of ex¬ ogenous GH causes a rise to normal of serum SM-A levels.5'"'5 High levels of SM-A, greater than 2 U/mL, are pres¬ ent in acromegaly, where the SM-A level has been found to express the activity of the disease better than does the GH level."4' High levels are also associated with chronic renal insufficiency and uremia, with no change being found after hemodialysis but a small decrease being evident after kidney transplantation.56 As yet, we have very little informa¬ tion on the effect of purified somatomedins. In one study, it was found that the injection of partially purified SM-A into hypophysectomized rats caused an increase of about 30% in tibial width although no increase in body weight was registered.57 Prelimi¬ nary experiments showed that the injection of serum peptide fractions containing SM into Snell dwarf mice resulted in acceleration of growth.58 Somatomedin-C

Somatomedin-C is

a

basic

peptide

arginine residues, with at least one sulfydryl bridge, with an isoelectric point at a pH of 8.4 to 9.2, and with a molecular weight of 7,600 daltons. Two separate electrophoretic peaks have been obtained in the final rich in

isolation. This peptide is closely related to SM-A, sharing all of its characteristics as an insulin-like activator and also promoting growth. Like SM-A, SM-C has specific receptor sites on cell membranes."" Furlanetto et al'" re¬ '"

cently reported for SM-C.

a

radioimmunoassay

Somatomedin-B

Somatomedin-B is an acidic peptide with an isoelectric point at a pH of 5.9 to 6.4 and a molecular weight of 5,000

daltons that is cross-linked by four disulfide bridges with TV-terminal aspartic acid. Analysis of the primary structure of SM-B shows that it is a unique protein not related to insulin or any of the other known growth factors.62 It is not possible to measure the purified form of this factor with any of the cartilage bioassays or bind¬ ing assays.63 Its biological activity is expressed in the proliferation of noncartilaginous cells, such as human embryonal lung fibroblasts and glial cells,61 and is measured by the stimu¬ lated rate of thymidine incorporation into DNA. An in vitro antiproteolytic activity has also been attributed to this peptide.6' Yalow et al's recently developed a radioimmunoassay for SM-B, showing high levels in the sera of acromegalics and low levels in hypopituitary dwarfs. There is a grad¬ ual decline in serum levels of SM-B with age65 and during estrogen thera¬ py.'16 During pregnancy, there is a rise that peaks at delivery. SomatomedinB has also been shown to be in human OTHER SOMATOMEDINFAMILY FACTORS Nonsuppressible, Insulin-like

Activity (NSILA) The nonsuppressible, insulin-like activity factor is a family of two or more peptides isolated from serum fraction Cohn III (precipitate B) (blood plasma proteins and some glob¬ ulins resulting from fractionation of whole plasma according to Cohn's

method) that has the property of not being suppressed by insulin anti¬

bodies and that possesses approxi¬ mately 90% of the total insulin-like activity of serum.67 Recently, NSILA has also been termed IGF (insulin-like growth factor).68 Two forms are known: a soluble form (NSILA-S), and an insoluble, precipitated form (NSILA-P).6" The latter is a high molecular weight peptide weighing about 90,000 daltons and suspected to be an artifact of the acid-ethanol treatment used in the purification procedure.69·70 The NSILA-S form is comprised of two low molecular weight peptides, NSILA-I (IGF-I) and NSILA-II (IGFII), both of which have a molecular weight of 5,800 daltons and consist of a single-chain molecule containing

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two disulfide bridges and a free SH group and lacking histidine (which is

present in SM-A).71 The NSILA-S has

isoelectric point in the range of a pH of 7.5 to 8.5 and is more basic than SM-A though less basic than SM-C. The first 30 residues have a striking an

resemblance to the beta chain of insu¬ lin,68 and the residues at sites 42 to 62 resemble the alpha chain of insulin,72 but there is no evidence for any simi¬ larity with the SM-C peptide region. The NSILA-S factor is found in serum in association with a specific high molecular weight protein (200,000 dal¬ tons) composed of several oligomers

weighing 50,000

to

70,000 daltons.7374

The NSILA-S carrier

protein is a /3-globulin; (association between sev¬ eral identical monomers) some inves¬ tigators identify it as the transferrin molecule.75 The formation of both NSILA-S and its carrier protein is

GH-dependent.76 The complex of NSILA-S carrier protein is biologically

and its inactive

insulin-like agent or as a growth promoter in vitro as well as in vivo. There are specific receptor sites on 78 many cell types for NSILA-S77 and also with insu¬ can NSILA-S compete lin for insulin-specific receptors, al¬ though its binding to such an insulin receptor does not initiate or stimulate any metabolic events. It is commonly agreed that free NSILA-S is the biologically active form in vitro and in vivo,7" although some reports claim that NSILA-S sulfation activity on cartilage and its insulin-like activity may also be initiated by the complex form. The NSILA-S has been found to have an insulin-like activity on the epididymal adipose tissue and the isolated fat cells of rats, as well as on muscle such as the diaphragm of the rat. It is not clear whether the sulfa¬ tion activity attributed to NSILA-S is an intrinsic characteristic, since stud¬ ies of highly purified preparations show that sulfation activity is depend¬ ent on the presence of another serum factor.8" In contrast to SM-A and SMC, NSILA-S acts as a growth stimula¬ tor on noncartilaginous cells, as shown by its stimulation of thymidine incor¬ poration by human fibroblasts in culture and its acceleration of their rate of cell division. Also reported has been a mitogenic effect of NSILA-S as an

chick embryo fibroblasts.81 As to the mode and route of action of NSILA-S, it is believed to inhibit adenyl cyclase; in fat and liver cells, it has been reported to inhibit epinephrine-induced lipolysis and the glucaon

gon

response.7"

The most common assays for deter¬ mining NSILA-S in purified form (at least one-step column on dextran gel filtration [Sephadex G-75] yielding about 100-fold purification) include: (1) a bioreceptor assay that uses membranes of rat fat-pad tissue, of human transformed lymphocytes, of chick embryo fibroblasts, or of rat liver cells82; (2) an NSILA-S binding protein assay83; and (3) radioimmu¬ noassay for IGF-I (according to a letter from Dr J. Zapf and Professor R. Froesch, in July 1978). In subjects without growth disor¬ ders, levels of NSILA-S were found to be about 100 µ /mL; in acromegalie subjects, the level was about 200 /xU/ mL; and in pituitary dwarfs, the level was as low as 37 µ /mL.8185 In studies of other types of growth disorders, normal values have been registered. High levels of NSILA-S have been noted in pregnant women, suggesting a possible role in fetal growth.86-87 Studies of carrier protein levels have shown levels of 330 µ /mL in subjects without growth disorders, 477 µ /mL in acromegalie subjects, 183 µ /mL in pituitary dwarfs, and 23 µ /mL in Laron-type dwarfs.88 Patients with liver cirrhosis were found to have levels of 85 µ /mL, whereas patients with nephrotic syndrome had normal levels.88 The administration of GH to pituitary dwarfs has been found to lead to an increase in levels of NSILA-S or IGF-I as well as to an increase in the insulin-like and sulfa¬ tion activities.81 Kastrup and Zapf8" reported an increase to normal of sulfate incorporation when purified NSILA-S was added to serum ob¬ tained from a Laron-type dwarf. Normal levels of NSILA-S have been measured in diabetic animals"" and in patients with uncontrolled diabetes,7" which supports the general belief that NSILA-S does not play an active role in the regulation of blood glucose."' However, NSILA-S has also been reported to increase in association with an elevation in blood glucose

levels. Only in patients with nonislet cell tumor, which generates NSILA-S, have hypoglycémie values been found."2 "' In dogs subjected to pancreatectomy, NSILA-S levels were found to drop drastically to zero lev¬ els and then to reappear after insulin therapy, a finding that sug¬ gests that insulin influences the level of NSILA-S.84"1"4

Multiplication-stimulating Activity Factor (MSA)

Attempts to isolate the active frac¬ tions of calf serum, which is the most common biological fluid used for promoting cell growth in culture, yielded a growth factor differing slightly from both NSILA-S and somatomedin, called multiplicationstimulating activity factor."5 This fac¬ tor can be isolated either by a proce¬ dure similar to that used for the puri¬ fication of NSILA-S and of SMs or by direct chromatography of whole se¬ rum over a cationic ion exchange resin that retains the biologically active agent. The product, which has a molec¬ ular weight of 4,000 to 5,000 daltons, is a highly active growth-promoting sub¬ stance that in studies on fibroblasts of chick and rat embryos as well as on human diploid fibroblasts has been found mainly to stimulate prolifer¬ ation of the fibroblasts and their synthesis of DNA. In its effects on DNA synthesis and in other proper¬ ties, it closely simulates the activities of SM-C, SM-A, and NSILA-S."""7 It is believed that MSA is also controlled by growth hormone, as are other fibroblast-stimulating factors."7 Its activity is measured by receptor assays for insulin-like action such as the human placental receptor assay for SM-A and SM-C and adipose receptor assays for NSILA-S. Evi¬ dence indicates that there are recep¬ tors for MSA in the membranes of cells of different tissues, eg, the liver, the placenta, fibroblasts, and trans¬ formed lymphocytes (which can also bind the somatomedins), NSILA, and even insulin."" These receptor studies emphasize the similarities between these growth factors. Recently, mole¬ cules similar to MSA but with higher molecular weights (around 10,000 dal¬ tons) have been isolated from cells derived from rat liver tumor and from

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platelets.1""1"1 These substances share common properties with the SMs and with fibroblast growth factor (to be discussed later).

S and S Proteins

From fetal calf serum, which is considered superior to calf serum in its ability to promote the growth of cells in culture, Hoffman et al'"2 have isolated two additional growth fac¬ tors, St and S2, that also stimulate the growth of embryonic rat fibro¬ blasts.1"21"3 Possessing a high molec¬ ular weight (620,000 daltons), S, has been shown to be a complex of a>macroglobulin and insulin that is active only when both components are present. S2 is an acidic protein with an isoelectric point at a pH of 4.9 and a molecular weight of 26,000 daltons. It does not cross react immunologically with S,. Acting together on the same cells, S, and S, generate a synergistic response.

GROWTH FACTORS ISOLATED FROM TISSUES In addition to the SM family of growth factors, a variety of growthregulating substances have been pro¬ posed by various investigators. Not all

the factors to be described have been chemically characterized and it cannot yet be said with certainty which of these agents are of physiological and/ or pathological importance in man. Nerve Growth Factor

(NGF)

Nerve growth factor is a protein molecule so-named because of its potent growth-promoting effect on embryonic sensory nerve cells as well as on fully differentiated sympathetic nerve cells."" It has been purified from mouse salivary glands1"5 and some of its precursor subunits (alpha, beta, and gamma chains) have been isolated.'"6 It has been found to be composed of a dimer of two identical peptides, each consisting of 118 amino acids with a molecular weight of 13,260 daltons and containing three disulfide bonds.'"71"8 Other sources of NGF or NGF-like substances have been snake venom, granuloma tissue, chick embryo tissues, L-cells (a human embryonic lung cell line), and 3T3 cells (a Swiss albino mouse embryonic fibroblast-like cell line).'""

The NGF is determined say that

uses

sensory

by a bioas¬ ganglia from

8-day-old chick embryos incubated in vitro as expiants for 12 to 16 hours. The growth effect is estimated on the basis of the halo-like outgrowth of the neurites, which expresses the density of the neurites and is graded on a scale from 0 to 4. The availability of sensitive in vitro bioassays""'" and radioimmunoassay of NGF subunits"2 have made possible the detection of this factor in cell-free extracts and the blood of different vertebrates, includ¬

ing man. Injections of NGF into a chick embryo cause a great increase in the number of sensory and sympathetic ganglions and in hyperinnervation of the viscera. Sensory nerve cells of certain ganglions are receptive to NGF only during a restricted period of their development, later becoming refractory. The vast majority of sym¬ pathetic nerve cells, however, respond to NGF from early inception to maturity, an exception being the adrenergic neurons that innervate the reproductive system in both sexes, which do not respond to NGF.'" Parasympathetic ganglions are refractory to NGF."3

Nerve growth factor was found to have no mitogenic effect on cultured fibroblasts. In vitro its main function seems to be prolongation of the sur¬ vival of nerve cells. The positive, pleiotypic effect of NGF is seen in tissue culture, in terms of neurite outgrowth and de novo synthesis of neurotubules that leads to the neurite extension. Nerve growth factor does not seem to act through alterations of cyclic aden-

osine-3', 5'-monophosphate (cAMP)

levels."17 In vitro studies indicate that NGF inhibits parathyroid hormone (PTH)-stimulated bone absorption by proteolysis of the PTH; this is most probably due to the gamma subunit of NGF, which simulates the action of

trypsin.1" The

occurrence of familial neuro¬ disorders involving hyperfunction of the sympathetic system or peripheral nerve-tissue overgrowth has led to studies of the NGF activity in man. Increased blood levels have been reported in patients with dis¬ seminated neurofibromatosis,"5 ·"" medullary carcinoma of the thyroid,"7

logic

and

Paget's disease of bone.111 Unex¬ pected was the recent finding of

normal levels of NGF but elevated levels of NGF subunits in the serum of patients with familial dysautonom-

ia,"3118 a comp]eXj autosomal, recessively inherited, neurologic syndrome characterized

by loss of nerve cells in sympathetic ganglions and impair¬ ment of sympathetic function. These

results will have to be further investi¬ any definite conclusions can be drawn as to whether the etiopathology of familial dysautonomia can be linked to an abnormality of the NGF. Further studies on NGF should be valuable in enlarging our knowl¬ edge of basic aspects of the neurosci¬ ences and also of inborn abnormalities and degenerative processes of the

gated before

nervous

system.

Epidermal (EGF)

Growth Factor

Epidermal growth factor is a singlechain polypeptide composed of 53 amino acids of known sequence"" that has a molecular weight of 6,045 daltons. Cohen isolated EGF from the submaxillary glands of adult male mice,'2" where it was found as a complex with the high molecular weight of 74,000 daltons. This complex is associated with a binding protein that in itself has no growth-promoting activity but which does show arginine esterase activity.121 The high degree of purification attained and the avail¬ ability of a sensitive radioimmunoas¬ say has made possible further investi¬ gation of this factor.122 It was found that the concentration of EGF in the submaxillary gland of the mouse was dependent on the level of androgens present.122

In addition to the molecule isolated from mouse tissue (m-EGF), a similar molecule having a lower molecular weight (5,300 to 5,500 daltons) that competes with m-EGF cell membrane receptors and antibodies has been found in human urine.123 Human /S-urogastrone and m-EGF share com¬ mon cell receptors and have identical regions of amino acid sequences, with a homology of about 80%.12112' The biological activity of EGF in vivo has been found to be manifested in the following diverse ways: through the enhancement of proliferation and

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keratinization of embryonic and neo¬ natal epidermis, through the stimula¬ tion of epithelial cell proliferation in organ cultures of embryonic skin and embryonic cornea of the chick and of the human fetus, and through the stimulation of the epithelial cells of mouse mammary glands and mamma¬ ry carcinomas.12613" In vitro, m-EGF has been shown to exert a mitogenic activity in various monolayered cul¬ tures of cells such as human fibro¬ blasts, HeLa, 3T3 cell lines, and other cell types in addition to epidermal cells.123131"135 It has been demonstrated that EGF acts by binding to the intact cell membrane and to cell homogenates causing an altered metabolic rate, eg, increased glycolysis."6 It is believed to accelerate the transport of small mole¬ cules, leading to an increased rate of synthesis of RNA and, in certain instances, of DNA as well. Recent studies indicated that it also activates phosphofructokinase."7 Cholera toxin, dibutyryl cAMP, and theophylline have all been found to counteract the action of m-EGF.138 Dexamathasone modulates the binding of EGF and its

activity.1-1" Epidermal growth factor alone, without its binding protein, is able to stimulate DNA replication as well as cell proliferation. In the form of its complex, which contains arginine es¬ terase, it has been found to have

a

synergistic effect. The biological ac¬ tivity of both EGF alone and of the EGF complex depend on the presence of a trace of serum, fetal calf serum being particularly effective in enhanc¬ ing this activity. In the presence of ascorbic acid or a matrix sheet of collagen, there is a partial additive effect obtained from the activity of the EGF or its complex, although this effect is less than that obtained with the addition of serum.123132 Each fibroblast from human fore¬ skin was found to bind about 10' mole¬ cules of EGF. No other peptidal hormone can compete with or displace EGF.128'3" Further studies in this direction may be facilitated by the recently described fluorescent assay for labeling EGF receptor sites.11" Clinically, it has been observed that EGF blocks the secretion of gastrin, and promotes the proliferation of

fibroblasts and the epithelization of the mucosa at the sites of experimen¬ tal ulcers.12412" Recent investigations using a homologous radioimmunoas¬ say111 showed that in normal men and women, the 24-hour urinary excretion of human EGF is 29.7 ± 1.1 and 39.8 ± 1.7 (m ± SE) µg/g of creatin¬ ine, respectively, whereas in patients receiving oral contraceptives, the amount excreted was much greater (60.1 ± 2.7 µg/g of creatinine). Fibroblast Growth Factor (FGF)

Gospodarowicz et al"2 identified an FGF in the bovine pituitary gland and brain"2"1 that proved to be a basic peptide with an isoelectric point at a pH of 9.5 and a molecular weight of 13,400 daltons.142·143 This proved to be a strong mitogen with a variety of human and animal cells, including fibroblasts, from amniotic fluid, mus¬ cle cells, rabbit ear cartilage, glia, the adrenals, bone, and cornea as well as endothelial cells from blood ves¬ sels."7"8 " "'-"2 When added in combina¬ tion with serum, FGF caused a reduc¬ tion in the doubling time of these cells."2 In a number of cultured fibro¬ blast strains (Balb/c 3T3 and 3T3), the mitogen potential and the ability to initiate DNA synthesis by FGF were less than that of serum; when FGF was tested in combination with glucocorticoids, however, its potential was found to be equal to that of whole serum.15' Virus-transformed fibro¬ blasts did not respond to FGF.'" Recent investigations showed that platelets also contain a high concen¬ tration of FGF.""·'55156 Platelet-de¬ rived FGF was found to exert biologi¬ cal action in vitro on chick fibroblasts and on cell lines of 3T3 fibroblasts as well as on arterial smooth muscle cells and mesodermal other tissue cells.152'53-'55-156 It is believed that FGF initiates a series of events that includes synthe¬ sis of RNA and of protein since it has been found that actinomycin-D and cycloheximide inhibit the effect of FGF, although there is no evidence supporting the involvement of cAMP.157 A radioimmunoassay devel¬ oped for human serum polypeptides with FGF activity showed that normal blood contains approximately 5 µg/

mL of this agent.1"1158 The presence of FGF in interstitial inflammatory fluid'5" suggests that FGF may be involved in the wound-healing pro¬ cesses of various tissues, although this requires further delineation.

Overgrowth Stimulating Factor (OSF) The proliferation of synovial fibro¬

blasts has been said to be stimulated

by an overgrowth stimulating factor (OSF)"1" as well as by a connective tissue activator described by Castor.15" These may be considered to belong to the same category as FGF, although this still remains to be delineated. Liver Growth Factor

(LGF)

Agents that have been found to stimulate the growth of liver cells have been designated "LGF" by us. Extracts from experimentally injured liver tissue have been found to stimu¬ late collagen biosynthesis."" Compari¬ son of the effect of extracts of gener¬ ating rat liver with those of resting liver on DNA synthesis and cell prolif¬ eration of hepatocytes showed the generating rat liver to have a higher content of growth stimulators.162"164 Basic tripeptides from human serum were found to prolong survival of normal liver cells as well as those of liver neoplasm'65; a synthetic tripeptide (glyhislys) has shown similar properties.""1

Ovarian Growth Factor

(OGF)

The observation that stable ovarian cell lines depend on the exogenous addition of gonadotrophins for growth and that crude pituitary preparations are more active than purified ones led to the isolation from the pituitary of a substance named "ovarian growth factor." This is a basic polypeptide with a molecular weight in the range of 10,000 to 13,000 daltons.1"71"" It was found that OGF is associated with a component of high molecular weight with which it forms a complex of 600,000 daltons. With various methods of in vitro treatment this complex disintegrates into 100,000 daltons of carrier molecules and 20,000 daltons of active OGF.17" The biological activity of OGF in vitro is clearly evident from its support of the growth of ovarian cells, but its

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physiological role

remains to be clari¬

fied.

Cartilage Growth

Factor

(CGF)

A number of factors have been isolated that enhance chondrogenesis and also increase the capacity for attachment and spreading as well as the length of survival and colony formation of cartilage cells.'7'177 These factors may be secreted by the cartilage tissue itself or by the pitui¬

tary.'71177 Recent reports suggested that CGF acts on membrane transport

and DNA synthesis.'78"18' The exis¬ tence of factors regulating bone formation, remodeling, and healing of fractures has also been postu¬ lated.182183 Chalones—Mitotic Inhibitors of Cells Chalones are substances that con¬ trol the mitotic activity of either the tissue in which they are produced or of neighboring tissue, inhibiting the growth process. This subject has recently been reviewed more exten¬ sively by Maurer.184 These substances are believed to be proteins or glycoproteins; their molecular weight ranges from 30,000 to 50,000 dal¬ tons.185187 The first chalone to be discovered was an epidermal factor found to be active only after its inter¬ action with epinephrine.185 Other such inhibitors that have been isolated include substances that inhibit carti¬

lage cells,'88"" mesenchymal cells,'"2 synovial fibroblasts,1"3 endothelial cells,1"1 and liver cells.1"5 The function

of most of these has been found to be associated with epinephrine.181 It is of note that chalones have relatively long

biologic half-lives.

CONCLUSION

The complex process of growth finds its expression in several forms: (1) linear growth, which mainly involves the skeletal tissue; (2) organ growth; (3) growth compensating for wear and tear; (4) regenerative growth compensating for massive pathological tissue loss, as in wound healing; and (5) compensatory growth of one element of a paired organ when the other has been damaged. In addi¬ tion to the well-established growthpromoting hormones such as the

thyroid hormones, pituitary growth

hormones,1"6 the sex hormones,1"7 and insulin,"18 a variety of substances

claimed to possess growth-promoting activity have aroused much interest in recent years. The most interesting among these

substances is the

family of SMs (including SM-A, SM-B, and SM-C, IGF I and II, and MSA), which may actually be classified as hormones. Should purified SMs become available in larger quantities so that clinical trials become possible, these sub¬ stances may eventually prove to be important therapeutic agents. Clinical observations strongly sug¬ gest the role of as-yet-undefined growth factors in a number of condi¬ tions. For example, in patients with cranio-pharyngioma, growth slows prior to operation but accelerates thereafter, despite a deficiency of pituitary hormones, including growth hormone.'""2"" It has been postulated that insulin is the hormone responsi¬ ble for linear growth in such chil¬ dren'"82'" but there is no convincing

evidence to support this and another factor may well be involved. Intrigu¬ ing possibilities are also suggested by the conditions of hemihypertrophy2"2 and macrodactyly.2"3 That the rela¬ tionship between the various growth factors may not be simple is indicated by the fact that SM activity has been found to be normal or even elevated in a number of disorders associated with short stature, such as gonadal dysgen¬ esis, hypoparathyroidism, and consti¬ tutional short stature. This suggests the existence of end-organ resistance to SM, a hypothesis that has also been brought forward to explain the short stature of pygmies, who have been found to have normal levels of both plasma HGH and SM.2"4 A field only

recently subjected to investigation promises to yield findings of particular interest is the possible role played by factors governing the growth of elements of the epidermis and that

and the nervous system. The purification of these factors and the consequent availability of sensitive methods of assay provide the scientific worker with an invaluable tool for the further investigation of this fascinating field. The possibilities for exploration are innumerable. It can be hoped that the achievements of

the the

coming years will answer many of questions and also provide us with

clinical aids in the treatment of children with growth disorders. new

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Growth factors.

Growth Factors Zvi Nevo, PhD, Zvi Laron, MD process of growth is still surrounded by an aura of mystery. The efforts of biologists the world over in...
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