Physiological Effects of Transforming Growth Factor ex

JAMES P. TAM

1. INTRODUCTION

27

From the conditioned medium of transformed cells, two types of mitogenic poly­peptides (ex and 13) known as transforming growth factors were isolated and char­acterized. Because of their origin and their abilities to confer phenotypic transfor­mation of cells in culture, much of the studies of transforming growth factors have been focused on their roles in malignancy and their relationship with the oncogenes (for a review, see Brown and Blakeley, 1984; Sporn and Roberts, 1985). However, because of the scarcity of these growth factors from natural sources, little is known about their physiological effects in animals. Recently, our laboratory has synthesized highly purified rat and human transforming growth factor (TGFex). These synthetic materials have been vigorously characterized and found to be indistinguishable from those obtained from the natural source. The availability of these growth factors has made it possible for the first time to study the physiological effects in whole animals. Here, we report our results using the synthetic TGFex.

1.1. Transforming Growth Factor a

Transforming growth factor ex (De Larco and Todaro, 1978a; Roberts et al .. 1980) is released extracellularly by tumor cells of various origins into the conditioned medium in culture. These include cell lines derived from established tumors and those from viral (Todaro et al., 1976; Kaplan et ai., 1982; Massague, 1983) or chemical transformation (Moses et al., 1981; Proper et al .. 1982). Transforming

JAMES P. TAM. The Rockefeller University. New York, New York 10021.

407

T. W. Moody (ed.), Neural and Endocrine Peptides and Receptors© Plenum Press, New York 1986

408 JAMES P. TAM

growth factor a is characterized by its ability to confer reversible phenotypic trans­formation of untransformed, normal indicator cells in culture. However, this trans­forming ability is modulated by transforming growth factor type ~ (TGF~). One of the most often used indicator cell lines are normal rat kidney (NRK) fibroblasts (De Larco and Todaro, 1978b). The NRK cells, when grown in the presence of both types of TGF, lose their density-dependent inhibition, assume transformed morphology, and overgrow in monolayer culture. Furthermore, NRK cells do not normally grow in soft agar, but in the presence of TGFa in culture, these cells acquire anchorage independence and respond to form colonies in soft agar. Since these properties are closely associated with transformation, they have led to the hypothesis that TGFa is produced as an autocrine growth factor by the transformed cells to sustain self-stimulation and proliferation (Sporn and Todero, 1980; Kaplan et al., 1982; Sporn and Roberts, 1985). More recent studies have shown that TGFa activity is found also in nonneoplastic tissue of embryonic (Proper et al., 1982; Twardzik et al., 1982b) and adult fluids, including serum, urine, and colostrum (Twardzik et al., 1982a). However, the results of these findings are neither con­clusive nor definitive, since the biological activities of TGFa are closely similar to those of another growth factor, epidermal growth factor (EGF), which is present in biological fluids and tissues in normal states, and since none of the functional assays can distinguish between these two growth factors unequivocally, the devel­opment of a specific antibody against TGFa may help to resolve these uncertainties.

1.2. Transforming Growth Factor 13

Transforming growth factor ~ is a homodimer of 22,000 daltons (Roberts et al., 1982; Sporn and Roberts, 1985). Unlike TGFa, TGF~ is found both in the conditioned medium of virally transformed cells in culture (Roberts et al., 1980; Anzano et al., 1983) and in normal tissues and physiological fluid. Blood platelets have been found to be a rich source of TGF~. Similarly, fetal calf serum also contains TGF~. The latter finding offers an explanation of the early confusions concerning the sole ability of TGFa in soft agar to induce colony formation. It is now clear that TGFa requires the presence of either TGF~ or fetal calf serum for the synergistic action of large colony formations. Transforming growth factor ~, when tested in NRK cells, has no mitogenic capacity, but recent studies have shown that TGF~ is a mitogen to other cell types. More interestingly, TGF~ also acts as an inhibitor to the growth and proliferation of certain cell lines in culture (Tucker et al., 1984). The biochemical actions of TGF~ are mediated through its own cell surface receptor, which is distinctly different from the EGF receptor. So far, it has not been shown that the TGF~ receptor possesses any tyrosyl-specific protein kinase activity. Undoubtedly, TGF~ represents a distinct growth factor inhibitor and does not belong to the EGF-TGFa family. A summary of the differences between TGFa and TGF~ is shown in Table I.

TRANSFORMING GROWTH FACTOR a

TABLE I. Comparison of EGF, TGFa, and TGFf3

EGF TGFa

Amino acids 53 50 pI 4.5 6.8 Peptide chain Single Single Precursor 1200 160 Soft agar growth Yes Yes Receptor EGF EGF Tumor cell No Yes

2. STRUCTURAL SIMILARITIES WITH EPIDERMAL GROWTH FACTOR

409

TGFf3

224

Dimeric

No TGFf3

Yes

The earliest indication that TGFa is structurally and functionally similiar to EGF derives from the observation that conditioned medium containing TGFs is capable of competing with EGF for the EGF receptor (Todaro et ai., 1976, 1980). Furthermore, Reynolds et ai. (1981) also found that TGFs from conditioned medium also induce EGF-receptor-associated protein kinase to autophosphorylation of its own receptor in a manner similiar to EGF. However, it is only recently that con­clusive evidence from sequence determination confirms that TGFa does share con­siderable structural and sequence similarities with EGF. However, it is also clear that sequence homology between TGFa and EGF is not as close as anticipated and that TGFa is distinctly different in sequence.

The primary structure ofTGFa from a retrovirus-transformed Fisher rat embryo fibroblast (rTGFa, Fig. 1), deduced from Edman degradation (Marquardt et ai., 1983, 1984; Massague, 1983) and the predicted cDNA sequence (Lee et ai., 1985),

50

FIGURE 1. Structure of rTGFa.

410 JAMES P. TAM

contains 50 amino acid residues (Fig. 1) and differs from that of the predicted cDNA sequence of the human TGFa in four amino acid residues (Derynck et al., 1984). Both the rat and human TGFa sequences share between 30 to 40% sequence homology with human (Gregory, 1975) and mouse EGF (Fig. 2). More interestingly, all four sequences can be aligned so that all six cysteine residues display homologous positions. Furthermore, amino acid residues (total four) related to conformational requirements for J3-bends such as glycine and proline can also be aligned to ho­mologous positions. Thus, it is likely that the sequence homologies between TGFs and EGFs serve to provide a common unique secondary structure for these growth factors to elicit binding to the same receptor. From the evolutionary point of view, it is also interesting to note that the sequence of TGFs is better conserved than EGFs between species. The sequence homology of TGFa between rat and human is 92% (46 out of 50 residues), but EGF between mouse and human is only 70% (37 out of 53 residues).

Another significant difference between EGFs and TGFas lies in their biosyn­thetic processing. The TGFas are derived from a precursor polypeptide of 160 amino acids, and the cleavage of the 50-amino-acid TGFa from the larger form occurs at alanine and valine residues at both amino and carboxyl termini. However, EGFs are derived from a precursor polypeptide of about 1200 amino acids, and the cleavage of the 53-amino-acid EGF occurs at paired basic residues, similar to those of other peptide hormones. Thus, structural comparison and biosynthetic processing suggest that TGFa is similar to but distinct from members of the EGF-growth factor family.

Another unexpected protein that shared similar sequence homology with TGFa is a peptide (residues 45-85) of a 140-residues polypeptide encoded by one of the early genes of vaccinia virus (Blomquist et al., 1984; Brown et al., 1985). Vaccinia viral peptide (VVP) shares 15 residues with both rTGFa and hTGFa and 19 residues with both mEGF and hEGF (Fig. 2). Thus, VVP is as similar to EGF as is TGF. More interestingly, several blood platelet factors, blood coagulation factor IX, factor

1 10 20

rTGFa VVSHFNKC PDS HTQYC FH-GTC RFLVQ hTGFa VVSHFNDC PDS HTQFC FH-GTC RFLVQ mEGF NSYPGC PSS YDGYC LNGGVC MHIES hEGF NSDSEC PLS HDGYC LHDGVC MYIEA VVP LC GPE GDGYC LH-GDC lllARD

30 40 50

EEKPAC VC HSGY VGVRC EHADL LA EDKP AC VC HSGY VGARC EHADL LA LDSYTC NC VIGY SGDRC QTRDL RWWELR LDKY AC NC VVGY IGERC QYRDL KWWELR IDGMYC RC SHGY TGmC QHVVL VDYQR

Common Sequence FIGURE 2. Sequence homology of TGFa, EGF, and VVP (vaccina viral

CxxxxxxCxxGxCxxxxxxxxxxCxCxxGxxGxxC protein).

TRANSFORMING GROWTH FACTOR ex 411

X, and other proteins such as low-density lipoprotein receptor and protein C also share distant sequence homology with the EGF and TGF family. The conserved sequence homology (as shown in Fig. 2), as the consensus sequence, mainly cor­responds to the cysteinyl and glycyl residues, which are important for the structural and conformational role of the molecule. The structural similarities of these peptides and growth factors may represent evolutionary conservation but may also indicate functional importance. Nevertheless, future experimental confirmation will be needed to settle this issue.

3. CHEMICAL SYNTHESES OF RAT AND HUMAN TGFa

One of the impediments in the study of TGFa is the difficulty to obtain pure and sufficient quantity of material from tissue culture. From 150 liters of the conditioned medium, only 1.5 I-lg of purified TGFa could be isolated. Consequently, none of the physiological studies has been carried out. In addition, the scarcity of TGFa also hampers the effort to prepare specific antibodies for the study of oc­currence and localization of TGFa in tissues. Our laboratory has recently undertaken the total synthesis of both rat and human TGFa with the objective of providing sufficient material to enable more extensive studies on their mode of action, par­ticularly in whole animals. Highly purified synthetic rat and human TGFas have been prepared by the solid-phase peptide synthesis method (Tam et ai .. 1984; Tam, 1985,1986; Sheikh and Tam, 1985). The homogeneity of these materials has been confirmed by reversed-phase high-pressure liquid chromatography (HPLC) (Fig. 3)

FIGURE 3. Results of HPLC analysis of synthetic rat (A) and human (8) TGFex. The arrow indicates the position where mEGF is eluted under the same conditions.

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412 JAMES P. TAM

and other physical characterization. Furthennore, rat TGFa coelutes with the natural TGFa as a single symmetrical peak under similar chromatographic conditions.

Both synthetic rat and human TGFas were compared with natural rat TOFa and mEGF for biological properties of the putative growth factor properties in vitro (Table II). In the mitogen assay, the stimulation of growth of serum-deprived nonnal rat kidney cells by the growth factor was measured by the incorporation of [1 251] iododeoxyuridine. In the soft agar assay and in the presence of TGFI3, the colony fonnation in soft agar was measured. Furthennore, the binding of these growth factors to the A-431 human carcinoma cells, whose cell surfaces are rich in EGF receptors, was also tested. As shown in Table II, all of the growth factors showed comparable activities and were active at the nanomolar level. A consequence of binding to the EGF receptor is the stimulation of the receptor-associated tyrosine protein kinase. Both EGF and TGFa were found to stimulate the phosphorylation of the exogenous synthetic angiotensinyl peptide substrate with similar half-maximal activities. These results support previous findings and provide convincing evidence that the synthetic TGFas are as active as the natural rTGFa and mEGF in vitro.

4. EFFECTS OF TGFa ON THE SOMATIC DEVELOPMENT OF NEWBORN MOUSE

Epidennal growth factor causes marked effects on somatic development when injected into a newborn mouse. Daily subcutaneous administration of microgram quantities accelerated tooth eruption and eyelid opening (Cohen, 1962) but retarded rate of body growth and inhibited hair growth (Cohen, 1962; Moore et ai., 1981; Panaretto et ai., 1982). To detennine whether TGFa possesses these physiological effects on newborn mouse, synthetic rat TGFa (0.05 to 4 fLg/g body weight) was injected daily subcutaneously into newborn NCS mouse to exmaine its effect on the somatic development.

TABLE II. Comparisons of Synthetic TGFa, Natural rTGFa, and EGF by Different Growth and Transforming Assays

Assay

Stimulation DNA synthesis EG F -radioreceptor Phosphotyrosine kinase Soft agar growth

" Natural rat TGFo:. b Synthetic rat and human TGF.

mEGF

1.4 1.7 0.1 0.6

Half maximal activity (nM)

nTGFaa sTGFab

1.7 2.3 3.7 4.2 0.4 0.3 0.5 0.3

TRANSFORMING GROWTH FACTOR 0: 413

4.1. Precocious Opening of Eyelid and Incisor Eruption

Early eruption of lower incisor teeth and opening of eyelids were observed in a dose-dependent manner with the TGFa-treated mouse compared with littermate controls (Fig. 4).

Doses lower than 0.25 f.Lg TGFa/g body weight produced no significant effects. At 2.7 f.Lg TGFa/g body weight, incisor tooth eruption and eyelid opening were observed on day 6 to day 7 instead of day 9 to 10 for tooth eruption and day 12 to 14 for eyelid opening seen in control animals. When EGF was compared to TGFa on both of these effects in a dosage of 2.7 f.Lg/g body weight, no significant difference was observed between TGFa and EGF. The eruption of upper incisors usually occurred a day after the lower incisor eruption. Thus, on day 8, eruption of both incisors of the EGF- or TGFa-treated mouse was clearly visible, whereas none was observed with the controls (Fig. 2).

4.2. Retardation of Hair Growth and Body Weight Gain

The growth rate of the first haircoat in male neonatal mouse has been found to be significantly retarded with daily injections of 4 f.Lg mEGF/g body weight for

6

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FIGURE 4. Dose-response curve of acceleration of precocious eyelid opening (e) and incisor eruption (_) in newborn mouse.

414 JAMES P. TAM

14 consecutive days from birth (Moore et at., 1981). More interestingly, infusing sheep intravenously with 0.12 mg mEGF/kg body weight results not only in depres­sion of wool growth but also in complete casting of the fleeces, leaving the sheep nude on the wool-growing regions of the body (Panaretto et at., 1984). Thus, at high doses, EGF has an inhibitory effect on DNA synthesis in the dermal skin sections that contain proliferation cells of hair or wool follicles. Furthermore, EGF also retarded rate of body growth (Cohen, 1962; Moore et at., 1981). Although the biochemical events leading to inhibiting effects have not been clearly established, the overall physiological effects nevertheless provide a source for comparison be­tween EGF and TGFa.

Similar to EGF (Carpenter and Cohen, 1979), treatment with TGFa also produced inhibition of hair growth correlating to dose administered (Table III). Examination of the overall rates of hair growth visually and by scanning electron microscope on glutaraldehyde-fixed skin at high dosages revealed that EGF or TGFa produced a significantly finer and shorter coat of montrichs compared to control animals. At 2.7 j.1g TGFal g body weight per day, approximately a 30% reduction of hair length and diameter was observed compared to control animals.

Transforming growth factor a was found to retard the overall growth rate of newborn mouse (Table II). The growth rates of the first 10 days of birth were examined, and in control animals, doubling and tripling of body weights were usually seen on days 5 and 9, respectively. However, the growth of the TGFa­treated animals was stunted at doses higher than 0.3 j.1g/g body weight per day. Again, the inhibition of rate of growth correlated well with the dose administered. At 4 j.1g/g body weight per day, the growth rates of either TGFa- or EGF-treated animals were 25% slower than the control animals.

Epidermal growth factor has been found to be of critical importance to the well-being of immature mouse during pregancy and the nursing period. This has led to the postulate that EGF may play an important role in physiological devel­opment and tissue differentiation. Sialoadenectomy of female mice decreases milk production and increases offspring mortality during the lactation period (Okamoto

TABLE III. Effect of Synthetic TGFa on Body Weight and Hair Growth on Newborn Mouse

Dose Mean (%)

(fLgig body weight Number of Treatment per day) animals Body weight Hair growth

Control No injection 22 100 100 or saline

EGF 22 2 75 63 TGF 0.5 20 112 107

0.5-1 8 94 87 1-3 6 85 76 4 3 76 65

TRANSFORMING GROWTH FACTOR ex 415

and Oka, 1984). It is possible that EGF or substances produced by EGF are lacking in sialoadenectomized mothers to pass along to the newborn mouse in milk during lactation. The similarities in physiological responses between TGFa and EGF are consistent with their role as growth factors and further suggest that TGFa is likely to be found in biological fluids during pregnancy and the nursing period.

5. STIMULATION OF ORNITHINE DECARBOXYLASE ACTIVITY

A remarkable physiological effect of EGF is its strong induction of ornithine decarboxylase activity in testes of neonatal mouse (Stastny and Cohen, 1970) and in the digestive tract (Feldman et at ., 1978). Since ornithine decarboxylase is a key enzyme in the biosynthetic pathway of the polyamines putrescine, spermidine, and spermine, whose production is closely related to mitogenic activities, the in vivo induction of TGFa will provide good evidence of the physiological similarity between these two mitogens. The level of the ornithine decarboxylase activity in a number of tissues was studied by subcutaneous injection of homogeneous synthetic TGFa or natural EGF into the 8-day-old mouse (Fig. 5). As a control, human growth hormone was also administered into the neonatal mouse under similar con­ditions . The responses elicited by TGFa or EGF from different tissues (three- to 24-fold) were found to be quite similar. A 24-fold increase in activity was seen in the testes by TGFa compared with a ten-fold increase by EGF. Significant but

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416 JAMES P. TAM

lesser increases (three- to fivefold) were also seen in the intestine and kidney. Little effect was observed in the spleen and brain.

When the time course of the induction by TGFu in these tissues was also examined, the decarboxylase activity in the testes reached a maximum in 4 hr and rapidly declined. However, the maximum level of induction in kidney was reached in 2 hr. Such responses were very similar to those effects elicited by EGF (Stastny and Cohen, 1970), which also caused a maximal response of ornithine decarboxylase activity in 4 hr.

To confirm that the induction of ornithine decarboxylase activity is accom­panied by protein synthesis, [3H]leucine was administered to the TGFu- or EGF­treated neonatal mouse. The effect ofTGFu or EGF on protein synthesis in different organs was examined by the incorporation of [3H]leucine into trichloroacetic-pre­cipitable proteins (Fig. 6). The results confirmed that TGFu induced new protein synthesis two- to tenfold compared with control. The greatest increase was seen in intestine (sixfold), and some increases were seen in spleen, kidney, and liver, but little effect was evident on brain, heart, or lung.

The marked but transient increase of ornithine decarboxylase activity is usually found in rapidly growing tissues or after the administration of growth-promoting hormones, peptides, or drugs. The elevated enzyme activity is accompanied by a net increase in polyamine synthesis and other cellular activities characteristic of growth and differentiation. In mammalian tissue, the highest concentration of poly­amine is found in the testes, and one of the most potent stimulators of ornithine

..,

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FIGURE 6. The TGFa stimulation of protein synthesis in tissues of an 8-day-old mouse.

TRANSFORMING GROWTH FACTOR Ct 417

decarboxylase in testes is EGF, which induces the enzymatic activity 20-fold in immature mouse. In this study, rat TGFa and EGF in similar dose level were found to stimulate the testicular ornithine decarboxylase activity of the immature mouse 24- and tenfold, respectively. Furthermore, the time course of the induction of enzyme activity by TGFa was similar to that by EGF. In comparison and under the same condition, human growth hormone produced only a threefold increase in activity. Thus, our findings put TGFa into the unique category of EGF, and no other peptide hormone known to date produced such a strong response of testicular ornithine decarboxylase stimulation as TGFa or EGF.

6. INHIBITION OF HISTAMINE-STIMULATED GASTRIC ACID SECRETION

Epidermal growth factor has been shown to inhibit gastric secretion stimulated by histamine, pentagastrin, or carbachol and insulin-induced hypoglycemia when given experimentally to animals. Recently, it has also been shown that an in vitro preparation of mammalian gastric mucosa from adult guinea pig is also a feasible model for the study of the inhibitory effects of EGF. However, the mechanism of this antisecretory activity is not known, but such anti secretory effect will provide another comparison for EGF and TGFa. When gastric mucosae were maintained in Ussing chambers in Ringer's solution, Murphy et al. (1985) and Rhodes et al. (1986) showed that TGFa was effective in the maximal reduction of the rate of secretagogue-induced acid secretion to the serosal gastric surface at a concentration of 120 ng of TGFa/m!. Similar maximal reduction of induced gastric secretion by mEGF in the matched tissue of the control experiment was also observed. The rate of inhibition was dose responsive and required 30 ng of TGFa/ml to be effective. Furthermore, both TGFa and EGF were found to produce the same electrophys­iological parameters following the growth factor treatments. These included in­creased resistance and decreased short -circuit current. However, both EGF and TGFa were ineffective as acid secretion inhibitors from the luminal side of the mucosa at the low doses that inhibited secretion from the serosal side. These data therefore suggest that TGFa behaves similarly to EGF in the secretagogue-induced acid secretion.

7. CONCLUSION

Although the biochemical events in the present studies of TGFa on newborn mouse remain unresolved, these results clearly support the results obtained from in vitro studies and provide the first direct in vivo evidence that TGFa is a member of the EGF family. In the present study, TGFa markedly accelerates precocious eyelid opening and incisor eruption and retards hair and weight growth in the newborn mouse. TGFa also stimulates ornithine decarboxylase activity and protein synthesis but significantly inhibits gastric acid secretion. These results also provide

418 JAMES P. TAM

a further stimulus for the search for the difference in the biosynthesis and physi­ological occurrence between TGFa and EGF. Unlike EGF, which is required to maintain the normal state of the animals, TGFa is found in states of rapid growth such as those found in pregnancy and malignancy and perhaps serves as the ad­ditional concurrent growth factor to meet such needs.

ACKNOWLEDGMENTS. This investigation was supported by PHS 36544 awarded by the National Cancer Institute, DHHS. I thank M. Sheikh, A. Nakhla, D. Rosberger, and L. Tsai for their contributions and technical assistance during the course of this work.

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