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Concise Review: Saliva and Growth Factors: The Fountain of Youth Resides in Us All
T. Zelles, K.R. Purushotham, S.P. Macauley, G.E. Oxford and M.G. Humphreys-Beher J DENT RES 1995 74: 1826 DOI: 10.1177/00220345950740120301 The online version of this article can be found at: http://jdr.sagepub.com/content/74/12/1826

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Concise Review

Saliva and Growth Factors: The Fountain of Youth Resides in Us All


T. Zelles1, K.R. Purushotham2, S.P. Macauley2, G.E. Oxford2, and M.G. Humphreys-Beher2*
'Department of Oral Biology, Semmelweis University Medical School, Budapest, Hungary; and the 2Department of Oral Biology, PO Box 100424, University of Florida, Gainesville, FL 32610; *to whom correspondence should be addressed

Abstract. The predominant focus of research dealing with saliva revolves around the role in the maintenance of oral health through a number of physiological and biological properties of constituent proteins. An ever-expanding literature exists indicating that the salivary glands additionally synthesize, and secrete into saliva, a wide range of growth factors. Animal studies with epidermal growth factor have provided evidence for a role in both oral and systemic health, through the promotion of wound healing rates. Thus, the ability to manipulate their rates of synthesis and absorption from saliva holds the potential to enhance tissue regeneration and homeostasis.

Introduction
Salivary constituents carry out a number of biological functions which are key to maintaining oral health. In fact, the component proteins, produced and released into the saliva by the salivary glands, are grouped according to their functions. These proteins aid in lubrication of the oral cavity (mucins, proline-rich proteins), remineralization (statherin, anionic proline-rich proteins), and digestion (amylase, lipase, proteases), as well as provide antimicrobial activity (proline-rich proteins, lysozyme, histatins, lactoperoxidase), and maintain mucosal integrity (water, mucins) (Herrera et al., 1988; Fox, 1989). However, missing from this last category, and in general discussions about saliva composition, is the incredibly rich source of growth factors that are present in saliva and their synthesis by the salivary glands. The healing properties of saliva were recognized as far back as 2000 years ago by the ancient Greeks, who applied snake saliva to open wounds to enhance cutaneous wound healing (Angeletti et al., 1992). It was the identification of epidermal growth factor from mouse submandibular glands (Levi-Montalcini and Cohen, 1960; Cohen, 1962) and its subsequent characterization that were recognized by the Nobel Award Committee for Medicine and Physiology in 1986. There is a large variety of polypeptides in saliva whose functions, in part, remain unknown. In addition, there are large gaps in the level of understanding of the properties of those growth factors that have been identified. These biologically active proteins include epidermal growth factor (EGF) and nerve growth factor (NGF), which are synthesized and secreted by the granular convoluted tubule cells (Barka, 1980; Gresik, 1980; Watson et al., 1985; Gresik, 1994). However, this short list of biologically active peptides produced by the salivary glands and released into saliva has grown significantly since the late 1980s to include transforming growth factor-alpha (TGF-oL: Yeh et al., 1989; Humphreys-Beher et al., 1994), insulin (Murakami et al., 1982; Smith and Patel, 1984; Kerr et al., 1995), insulin-like

Key words: salivary glands, epidermal growth factor, saliva, wound healing.

Received June 26, 1995; Accepted October 19, 1995

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growth factors I and II (IGF-I, IGF-II: Costigan et al., 1988; Ryan et al., 1992; Kerr et al., 1995), transforming growth factor-beta (TGF-P: Amano et al., 1991; Jaskoll et al., 1994), and fibroblast growth factor (FGF: Hiramatsu et al., 1994). These growth factors have been identified, for the most part, in animal and human saliva and animal salivary glands.

Expression and presence of growth factors in salivary glands


While the biosynthesis of most of these growth factors has been confirmed by in situ hybridization, immunohistochemical localization, reverse transcriptase-polymerase chain reaction, and in vitro synthesis using radiolabeled amino acids and biochemical purification, a degree of skepticism remains as to the origin of these proteins found in saliva. Primarily, it has been suggested that these compounds could potentially find their way into saliva via movement of serum macromolecules through the salivary glands (Vallejo et al., 1984; Fekete et al., 1993). Lending to this hypothesis is that stimulation of exocytosis in rat salivary glands by t-adrenoceptor agonists leads to a transient increase in epithelial cell tight-junction permeability (Hand, 1987). Macromolecular proteins in interstitial fluid or blood might gain access to the oral cavity by this mechanism. A second exo-salivary gland source of growth factors in saliva, derived from serum, could occur through the gingival sulcus. The overwhelming evidence, however, supports the concept that synthesis of growth factors is primarily performed by the ductal cells of salivary glands. Immunological detection and receptor-binding assays provide partial evidence for the synthesis of biologically active peptides in the case of EGF, TGF-oL, and insulin (Massague, 1983; Purushotham et al., 1995).

mice typically secreting a 100-fold higher level of growth factor into saliva than female mice (Byyny et al., 1974; Gubits et al., 1986). On the other hand, serum levels of EGF are similar in both sexes (Olsen et al., 1984; Kurachi and Oka, 1985). Genetic models of insulin-dependent diabetes mellitus as well as chemically induced diabetes have been shown to decrease the level of synthesis and thus lower saliva concentrations of EGF and NGF (Kasayama and Oka, 1989; Hu et al., 1992; Serrero et al., 1993). The decline of EGF synthesis in the salivary glands in diabetes could explain the poor rates of wound healing in this condition. Because diabetic animals develop peripheral neuropathy similar to that in humans, and NGF is known to promote sympathetic and sensory neuron development (Robertson and Sima, 1980; Scott et al., 1983), by analogy, these studies further suggest that the decreased production of NGF by the submandibular gland could potentially be involved in the pathogenesis observed in diabetic neuropathy (Kasayama and Oka, 1989).

Putative roles for epidermal growth factor (EGF)


Approximately 80% of the EGF present in mice is synthesized by the ductal cells of the submandibular gland (Rall et al., 1985). While producing less EGF than mice, the submandibular gland in rats is also the source of growth factor recovered from saliva (Olsen et al., 1984). In humans, both the parotid and submandibular glands appear to be the source of EGF found in saliva (Thesleff et al., 1988). Also, there is no evidence for the sexual dimorphism of expression in humans and rats as has been observed in mice. Epidermal growth factor has been implicated in a number of biologic processes outside of the oral cavity. EGF synthesized in the salivary glands and secreted into saliva appears to have a systemic effect on skin and gastric woundhealing processes in vivo (Niall et al., 1982; Olsen et al., 1984; Bodner, 1991). Furthermore, removal of the submandibular gland in animal models leads to ulceration of the intestinal mucosa (Skinner and Tepperman, 1981; Konturek et al., 1988). Cutaneous and oral wound healing as well as liver regeneration are significantly delayed by sialoadenectomy (Fisher, 1990; Noguchi et al., 1991a,b; Jones et al., 1995). EGF has additionally been shown to influence molar drift and orthodontic tooth movement (Dolce et al., 1994). In humans, there is a burst of EGF released into saliva following surgery for the removal of impacted molars (MacNeil et al., 1988). Removal of the submandibular gland in animals has been shown to reduce systemic organ homeostasis (Tsutsumi et al., 1986, 1993; Schneyer and Humphreys-Beher, 1988; Liu et al., 1994). Interestingly, sialoadenectomy has a potentially beneficial side-effect. Both mammary tumorigenesis and

Biological significance of growth factors present in sa iva


The main questions remain: Why are these growth factors synthesized and secreted into saliva and what are their functions? The oral mucosa is frequently a site of physical and chemical trauma from mastication, ingested foodstuffs, and pathological processes. The maintenance of its surface integrity is a property of the oral mucosa shared with skin and other mucosal surfaces with exposure to the external environment. Oral soft tissue follows a characteristic sequence of stages of wound repair which includes: (i) an inflammatory stage, (ii) a proliferative and repair stage, and (iii) a remodeling stage typical of most tissues. This repair process requires the presence of growth factors for proper rates of healing (MacKay et al., 1992). However, it is wellrecognized that the repair of oralsoft tissue is more rapid than that of cutaneous wounds. The most detailed example, in terms of potential function in wound healing processes, is EGF. Studies over the years on EGF suggest a role in the maintenance of oral and systemic health. Epidermal growth factor was first identified as a component from mouse submandibular gland extracts that induced precocious eyelid opening and incisor tooth eruption in neonatal mice (Cohen, 1962). In mice, the submandibular gland produces the highest level of EGF and NGF of any organ in the body (Rall et al., 1985). Synthesis of these growth factors in the submandibular gland is androgen-dependent, with male

chemically-induced hepatocellular carcinogenesis

are

reduced as a consequence of lower EGF levels caused by sialoadenectomy (Kurachi et al., 1985; Yamamoto et al., 1994).

Transforming growth factor-alpha (TGF-cx)


Transforming growth factor-alpha was originally isolated in in vitro cultures of virally transformed mouse cells. Although synthesized by many carcinoma cells, TGF-c- is also synthesized by a large variety of cells including hepatocytes, keratinocytes, and cells from the gastrointestinal tract, brain,

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Insulin is necessary for the active transport of glucose membranes. Diminished secretion of insulin results in hyperglycemia, glucosurea, weakness, and weight loss (Rossini et al., 1985). Diabetes also affects the general metabolism of protein and fat, leading to complications affecting the function of many organ systems, including the salivary glands. Insulin's effects on salivary gland homeostasis appear to be well-established in terms of regulation of amylase synthesis in acinar cells (Kim et al., 1990). Activation of the insulin receptor by ligand results in signal transduction through the phosphotyrosine secondmessenger pathway (White and Kahn, 1994). Chronic injection of mice with high concentrations of insulin induces parotid and submandibular gland hyperplasia through the activation of phosphotyrosine signaling components (Wang
across cell

and placenta (Derynck, 1988). TGF-ao shares structural similarity with EGF, and is a member of the EGF-like growth factor family. As such, it binds to the EGF receptor to exert its biological properties. The growth factor is synthesized by the ductal cells of the parotid and submandibular glands in rodents; however, unlike EGF, there is no evidence for a sexual dimorphism of its expression (Humphreys-Beher et al., 1994). The role of TGF-a- in maintaining epithelial cell growth and implication in wound healing are evidenced by transgenic mice in which TGF-a is overexpressed and subsequently causes epithelial cell hyperplasia in a number of organ systems (Vasser and Fuchs, 1991). Overexpression of TGF-ac and gastrin in the pancreas using an insulin promoter markedly increased duct cell metaplasia (Wang et al., 1993). Null mutants of TGF-a in transgenic mice, while appearing healthy and fertile, showed physical and histological alterations in skin, coat, and corneal formation (Mann et al., 1993).

et al., 1994a,b).

The fibroblast growth factor family (FGF)


Three proteins of the FGF family are thought to be important regulators of wound healing: acidic FGF, basic FGF, and keratinocyte growth factors. FGFs are potent mitogens for many types of endothelial, epithelial, mesenchymal, and neuronal cells (MacKay et al., 1992). An important characteristic of FGFs is their ability to bind to heparin and heparin sulfate. Immunohistochemical analyses of tissues for basic FGF often reveal an association with extracellular matrix and basement membrane. FGF stimulates proliferation of vascular cells, fibroblasts, and myoblasts. Additionally, FGF can promote cell migration, neovascularization, and formation of granulation tissue.

The detection of insulin in the parotid and submandibular glands and recognition of its synthesis by immunoassay and immunohistochemistry have been controversial (Carter et al., 1995). The apparent structure of salivary insulin is identical to that of pancreatic insulin. This has given rise to speculation that the source of insulin in saliva is the pancreatic islets, with blood transport responsible for its presence in the salivary glands and saliva. However, the presence of insulin mRNA and incorporation of radiolabeled amino acids into immunoprecipitable insulin in in vitro cultured parotid gland slices led to the conclusion of active synthesis of insulin by the salivary glands (Murakami et al., 1982; Kerr et al., 1995).

Nerve growth factor (NGF)


It has long been recognized that the salivary glands of rodents are a rich source of NGF as well as EGF (Bowcock et al., 1988; Murphy et al., 1993). However, the role of NGF in oral health or systemic health is questionable. Noguchi et al. (1991a) demonstrated that NGF did not promote wound healing of the tongue in contrast to EGF and TGF-a application. Alteration of NGF levels in various tissues following denervation or in vitro culturing has been taken as evidence of its important role in neuronal regeneration (Richardson and Ebendal, 1982). Transgenic mice expressing NGF through the leukemia inhibitory factor promoter in pancreatic cells showed enhanced sympathetic nerve innervation of the pancreas (Bamber et al., 1994). Studies by Schneyer and and Humphreys-Beher (1988, 1990) and Schneyer and co-workers (1992) showed that proliferation of parotid gland and exocrine pancreas acinar cells in the rat could be induced by the injection of NGF. The effect of NGF on proliferation appeared to be indirect, and resulted from increased glandular concentrations of norepinephrine owing to increased sympathetic innervation.

Insulin and the insulin-like growth factor family (IGF-I and IGF-II)
Insulin-like growth factors I and II have substantial amino acid sequence homology to pro-insulin (Baxter, 1986). Both IGFs are synthesized as large precursor molecules which are proteolytically cleaved to release the biologically active monomer. While pro-insulin requires further processing to remove the C-peptide for generation of biologically active insulin, IGF-I and IGF-IJ retain this segment, which is required for biological activity. The activities of insulin, IGFI, and IGF-II are modulated by specific binding proteins present in serum (Dai et al., 1994). Insulin-like growth factors I and II are synthesized in a wide variety of adult tissues, with IGF-I synthesis primarily occurring in the liver, heart, lung, kidney, and brain. IGF-II synthesis occurs predominantly during fetal development and in tumor cells of the liver (Rogler et al., 1994). The duct cells of the submandibular and parotid glands of mice and rats synthesize both IGF-I and IGF-II (Kerr et al., 1995). Both IGF-I and IGF-II have been isolated from human saliva (Costigan et al., 1988). Transgenic mice overexpressing IGFII develop hypoglycemia and hypoinsulinemia. IGF-I is a potent regulator of oligodendrocyte development in the brain and myelination of the central nervous system (McMorris et al., 1993). Transgenic mice overexpressing IGFI demonstrate the capacity of IGF-I to regulate brain growth (D'Ercole, 1993).

Transforming growth factor-beta (TGF-,) family


The transforming growth factor-beta family consists of TGF111-3, the bone morphogenic proteins (BMP)1-6, and a host of other factors. Unlike other growth factors, all members of the TGF-S family act as potent inhibitory compounds for a wide variety of cell types (MacKay et al., 1992). TGF-fs are synthesized by a wide variety of cell types, including platelets, macrophages, lymphocytes, fibroblasts, bone cells,

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and keratinocytes. The BMPs were originally identified by their presence in bone-inductive extracts (Butler et al., 1977). BMPs can induce cartilage and bone formation through osteoblast differentiation when implanted ectopically into rats (Wozney et al., 1990). High levels of expression for BMPs have been reported in developing fetal tissues, including limb buds, hair, heart, and teeth, leading to the suggestion of a critical role in morphogenesis that appears to be a characteristic of the TGF-19 super-family (Lyons et al., 1990). While saliva-derived EGF may be cytoprotective against gastrointestinal tract ulceration by promoting mucosal epithelial cell replacement, purified recombinant TGF-fl has been shown to suppress growth of certain cancer cell lines in vitro. Human colon cancer subsets have recently been identified with defects in mismatch repair genes that lead to a specific loss of the type II TGF-fg receptor. This strengthens the argument for a potential role for TGF-I in regulating normal colon cell function (Markowitz et al., 1995). Thus, it is not unrealistic to speculate that salivaderived TGF-1 may be important in maintaining overall health of the digestive tract.

The oral application of growth factors such as EGF for the treatment of gastric ulcers has been proposed based upon the ease of ingestion and direct delivery (Itoh et al., 1988). While growth factors in general demonstrate acid-resistant degradation, oral delivery is still subject to problems of accurate dosing. Uptake through the gastrointestinal tract leads to a short half-life, principally due to portal vein release into the hepatic system (Covelli et al., 1972; Kim et al., 1988). Alternatively, administration of polypeptide hormones has been investigated for rectal, vaginal, buccal, nasal, ocular, and transdermal uptake and dissemination (Fisher, 1923; Anders et al., 1983; Chiou and Chuang, 1988).

Is saliva a "fountain of youth?"


Are the healing properties of saliva interms of improved oral and systemic health real or imagined? The ancient Greeks were the first to demonstrate the healing powers of saliva through the application of non-venomous snake saliva to enhance cutaneous wound healing. In 1962, this property was rediscovered by the isolation of EGF from salivary glands and application of neonatal mice to promote precocious incisor tooth eruption and eyelid opening. Substantial work remains to increase our understanding of how saliva-derived growth factors can act systemically in an endocrine-like fashion. The evidence accumulated over the last 30 years supports this potential for EGF. Insulin, IGF-I, and IGF-II are commonly associated with specific binding proteins that modulate their effective interactions with receptors on the target cell surface. Are these same proteins in saliva? Do they protect bound growth factors from degradation in the gastrointestinal tract? More fundamentally, it remains to be answered whether many of the growth factors present in saliva are synthesized and secreted as biologically active peptides and why, since so many cell types seem capable of their own synthesis. Epidermal growth factor and TGF-o isolated from saliva possess the ability to bind to receptors in radioreceptor binding assays (RRAs), thus indicating that they are released as mature proteins. Several conditions appear to contribute to the effectiveness of growth factor treatment of wounds. One important consideration is that many growth factors require prolonged exposure to wounds to stimulate cell commitment to proliferation. In the oral cavity and gastrointestinal tract, the presence of a mucin coating at the epithelial/environmental interfaces may act to capture saliva-derived growth factors in the same manner as heparin acts in the extracellular matrix to confine FGF. Combinations of growth factors may be important in the optimization of acceleration of wound healing. Finally, another consideration may be the need for protease inhibitors in the application of growth factors for enhanced wound healing (Wysocki et al., 1993). In this regard, protease inhibitors present in saliva, such as the cystatins or histatin 5, may serve this purpose for growth factor activity. With the continued interest of the dental research community in salivary constituents, unique delivery systems or pharmacological/molecular manipulations of salivary gland protein synthesis may provide new therapeutic opportunities for the treatment of both oral and

Possible therapeutic applications and limitations


The uptake and distribution of growth factors from saliva that allow for systemic distribution and tissue influence are other areas of growth factor physiology where our knowledge is limited. Thornburg and co-workers (1984, 1987) have shown that there is a decline in gastrointestinal absorption with increasing age in the rat. Additionally, gastrointestinal uptake through the portal vein leads to destruction of growth factors by the liver (Kim et al., 1988). The outflow of gingival crevicular fluid into the oral cavity limits the topical application of growth factors to aid in periodontal regeneration (Lynch et al., 1991). Thus, the therapeutic use of growth factors for oral wound healing is limited by this approach. Where growth factors can be confined to the area of interest, as exemplified by studies on dentin/pulp chamber regeneration with members of the TGF-t growth factor family, there is evidence to support their potential therapeutic value (Vainio et al., 1993; Nakashima, 1994; Shirakawa et al., 1994). Recently, our own studies have shown a potential second route of uptake for growth factors from saliva, which also leads to systemic distribution (Purushotham et al., 1995). Confinement of EGF to the sublingual vasculature of rats leads to the rapid absorption and systemic tissue distribution of biologically active EGF in the rat. High concentrations of growth factor were noted first in the head and neck tissue, followed by organs distal to the head. A sublingual delivery system may prove to be advantageous for the introduction of growth factors for therapeutic uses in the oral cavity as well as systemically. The potential to harness the array of growth factors present in saliva is dependent upon receptor distribution on oral and other tissues and the ability to manipulate their ligand concentrations in saliva. The therapeutic use of growth factors and hormones in the treatment of a variety of human pathologies is presently hampered by the design of appropriate delivery systems.

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systemic diseases, thus maximizing the use of this regenerative fluid within us all.

Acknowledgments
This work was supported by NIDR grant DE 08778 to MHB, DE 10234 to KRP, and the Health Science Council grant T-02 352/93 and the Hungarian National Foundation for Scientific Research (OTKA) No. 1083 to TZ. GEO and SPM are supported by the Oral Biology Training Grant DE 07200.

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