DEVELOPMENTAL DYNAMICS 212:346–351 (1998) Tooth Eruption Molecules Enhance MCP-1 Gene Expression in the Dental Follicle of the Rat BENITO G. QUE AND GARY E. WISE* Department of Veterinary Anatomy and Cell Biology, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana ABSTRACT Tooth eruption is a localized developmental event that requires the presence of the dental follicle, a loose connective tissue sac that surrounds each tooth. Early postnatally in the first mandibular molar of the rat there is an influx into the follicle of mononuclear cells (monocytes) which, in turn, fuse to form osteoclasts that resorb the bone to form an eruption pathway. The chemoattractant that may attract the mononuclear cells to the follicle to initiate the cellular events of eruption is monocyte chemotactic protein-one (MCP-1). MCP-1 is secreted by the dental follicle cells and its gene is expressed maximally at an early postnatal age, correlating with the monocyte influx into the follicle. In this study, we show that other potential tooth eruption molecules—EGF, IL-1a, TGF-b1 and CSF-1—all enhance the expression of the MCP-1 gene in the cultured dental follicle cells. In vivo, injections of IL-1a or EGF also enhance the gene expression of MCP-1 in the follicle with maximal enhancement occurring in the early postnatal days. Thus, there appears to be a redundant function of the different tooth eruption genes to ensure that the MCP-1 gene is expressed. In turn, expression of MCP-1 may be critical for recruiting the monocytes to the dental follicle to initiate the cellular events of tooth eruption. Dev. Dyn. 1998;212:346–351. r 1998 Wiley-Liss, Inc. Key words: dental follicle; MCP-1; tooth eruption INTRODUCTION The initiation of tooth eruption involves an influx of mononuclear cells (monocytes) into the dental follicle with a concurrent increase in osteoclasts on the alveolar bone (Marks et al., 1983; Wise and Fan, 1989; Cielinski et al., 1994). The osteoclasts appear to be formed from the monocytes (e.g., see Wise et al., 1985) and their resorption of alveolar bone is required for eruption to occur (see Marks et al., 1994). Osteopetrotic mutant rodents are deficient in osteoclasts and, as such, have formed but unerupted teeth (Cotton and Gaines, 1974; Iizuka et al., 1992; Grigoriadis et al., 1994). Bone resorption also can be reduced by injection of bisphosphonates and this inhibits the rate of tooth eruption (Grier and Wise, 1998). r 1998 WILEY-LISS, INC. Recently, we have shown that two molecules secreted by rat dental follicle cells, colony-stimulating factor-one (CSF-1) and monocyte chemotactic protein-one (MCP-1), are chemotactic for mouse monocytes in vitro (Que and Wise, 1997). Both genes are maximally expressed in the rat dental follicle at the time of maximal influx of monocytes into the follicle (day 3 postnatally) (Wise et al., 1995a; Que and Wise, 1997). In turn, CSF-1 expression is enhanced by a cascade of molecular signals (Wise and Lin, 1995). Specifically, interleukin-one alpha (IL-1a) will enhance CSF-1 expression both in vitro (Wise and Lin, 1994) and in vivo (Wise, 1998). IL-1a itself is enhanced by either epidermal growth factor (EGF) or by transforming growth factor-beta one (TGF-b1) (Wise et al., 1994). The effect of these putative tooth eruption molecules on MCP-1 gene expression in the dental follicle is unknown. Thus, it was the aim of this study to determine the effect of EGF, TGF-b1, IL-1a and CSF-1 on the expression of MCP-1 in cultured dental follicle cells. In addition, in vivo studies were conducted to determine whether injections of EGF or IL-1a would enhance MCP-1 expression in the dental follicle at different days postnatally. Because the presence of the dental follicle is required for eruption to occur (Cahill and Marks, 1980; Marks and Cahill, 1984), elucidation of the molecular signals that might be required to recruit monocytes to the follicle is critical for understanding the relationship between the molecular and cellular events of tooth eruption. RESULTS All of the potential tooth eruption molecules examined (EGF, TGF-b1, IL-1a and CSF-1) enhance the gene expression of MCP-1 in the cultured dental follicle cells both time- and dose-dependently. TGF-b1 has a dramatic effect on MCP-1 expression, resulting in peak expression at 3 hr after incubation with a decline at later times (Fig. 1). A dose response study indicates that incubating the cells in TGF-b1 at a concentration of 2 ng/ml medium elicits the maximal increase in gene expression with a slight decline at higher doses (Fig. 2). Grant sponsor: National Institute of Dental Research; Grant number: DE08911. *Correspondence to: Dr. Gary E. Wise, Department of Veterinary Anatomy and Cell Biology, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803-8408. Received 10 October 1997; Accepted 2 February 1998 MCP-1 EXPRESSION IN THE DENTAL FOLLICLE Fig. 1. Ethidium bromide-stained gel of RT-PCR product for MCP-1 in dental follicle cells after incubating the cells in TGF-b1 at a concentration of 1 ng/ml medium for different times. Note that the maximal enhancement of MCP-1 gene expression is after 3 hr incubation in TGF-b1 (lane d) with a subsequent decline at later times (lanes f, g). Lane a, molecular weight standards. Lane b, control, no TGF-b1. As seen in lower portion of figure, b-actin mRNA levels are not affected by TGF-b1. Fig. 2. Gel of RT-PCR product for MCP-1 after follicle cells were incubated for 3 hr in TGF-b1 at different concentrations. Peak enhancement of MCP-1 expression appears to be at a concentration of 2 ng TGF-b1/ml medium (lane e). Lane b, control. Fig. 3. Gel of RT-PCR product for MCP-1 after follicle cells were incubated in EGF at a concentration of 10 ng/ml for different times. Maximal expression of MCP-1 gene expression appears to be at 3 hr incubation in EGF (lane d). Lane b, control. Regarding EGF, the optimal time of incubation for maximal expression was 3 hr (Fig. 3) in a time-course study. In a concentration-dependent study, doses of EGF at 5–250 ng/ml increased the gene expression of MCP-1 over the control (Fig. 4) with the maximum being reached at 25–50 ng/ml (Fig. 4). IL-1a also enhances MCP-1 transcription time-wise, with a maximal expression at approximately 3 hr and then a decline at 24 hr (Fig. 5). In terms of a dose response, it appears that 25–50 ng/ml is the optimal amount for increasing MCP-1 expression (Fig. 6). The effect of CSF-1 on MCP-1 expression is similar to the other molecules in that a 3-hr incubation is the optimal time for maximal expression of MCP-1 (Fig. 7). The optimal dosage of CSF-1 for MCP-1 transcription is approximately 40 ng/ml (Fig. 8). 347 Fig. 4. Gel of RT-PCR product for MCP-1 after follicle cells were incubated for 3 hr in EGF at different concentrations. Concentrations of 5 to 250 ng of EGF increased, MCP-1 expression (lanes c–g) with the peak being reached at 25–50 ng/ml (lanes d, e). Lane b, control, no EGF. Fig. 5. Gel of RT-PCR product for MCP-1 after follicle cells were incubated in IL-1a at a concentration of 1 ng/ml for different times. Maximal expression of MCP-1 is at 3 hr (lane d) with a subsequent decline at later times (lanes e, f). Lane b, control, no IL-1a. Fig. 6. Gel of RT-PCR product for MCP-1 after follicle cells were incubated in IL-1a for 3 hr at different concentrations. Concentrations of 5 to 250 ng of IL-1a/ml medium increased MCP- 1 expression (lanes c–g) with the peak being reached at 25–50 ng/ml (lanes d, e). Lane b, control. In vivo, the potential tooth eruption molecules also enhance the gene expression of MCP-1 in the dental follicle. Injections of EGF at days 1, 3, 5 and 7 increase the expression of MCP-1 with the greatest increases occurring at either day 1 or day 3 postnatally (Fig. 9). Injections of IL-1a and examination of MCP-1 mRNA at days 3, 7 and 10 show that MCP-1 gene expression is enhanced at each age after IL-1a injection, with the greatest increase seen at day 3 (Fig. 10). DISCUSSION A fundamental tenet of tooth eruption is that alveolar bone resorption must occur to form an eruption pathway (e.g., see Marks et al., 1994; Grier and Wise, 1998). To achieve this, there is an influx of mononuclear cells (monocytes) into the dental follicle to increase the 348 QUE AND WISE Fig. 7. Gel of RT-PCR product for MCP-1 after follicle cells were incubated in CSF-1 at a concentration of 10 ng/ml for different times. Maximal expression of MCP-1 is seen at 3 hr (lane d) with a slight decline at later times (lanes e–g). Lane b, control, no CSF-1. Fig. 8. Gel of RT-PCR product for MCP-1 after follicle cells were incubated in CSF-1 for 3 hr at different concentrations. Peak expression of MCP-1 is seen at a concentration of 40 ng CSF-1/ml (lane d) with a decline at higher concentrations (lanes e, f). Lane b, control. Fig. 9. In vivo analysis of MCP-1 expression in the dental follicle at different days postnatally. Animals were either injected with EGF (1) or not (-). As can be seen in ethidium bromide stained gel, RT-PCR product for MCP-1 is enhanced by EGF injection with the greatest increases occurring at day 1 and day 3. number of osteoclasts needed to erode the alveolar bone for eruption (Marks et al., 1983; Wise et al., 1985). In the rat, the maximal number of monocytes and osteoclasts in the first mandibular molar is reached at day 3 postnatally (Wise and Fan, 1989; Cielinski et al., 1994). For the above cellular events to occur requires the recruitment of the monocytes into the follicle. Recently, we have shown that MCP-1 is expressed in the dental follicle with its peak expression at day 3 postnatally (Que and Wise, 1997). Moreover, the medium taken from cultured follicle cells is chemotactic for monocytes, as is added MCP-1 only, and treating the medium with an antibody to MCP-1 reduces this chemotaxis (Que and Wise, 1997). Thus, it is probable that MCP-1 secreted by the dental follicle acts as a chemoattractant to recruit monocytes into the follicle for eruption. Another candidate chemotactic molecule, CSF-1, also is secreted by the follicle (Grier et al., 1998) and is chemotactic for monocytes, although MCP-1 is a more Fig. 10. In vivo analysis of MCP-1 expression in the dental follicle at different days postnatally. Animals were either injected with IL-1a (1) or not (-). As can be seen on the gel, the RT-PCR product for MCP-1 is enhanced by IL-1a injection with the maximal increase seen at day 3 (lane c). potent chemoattractant than is CSF-1 (Que and Wise, 1997). As seen in this study, the gene expression of MCP-1 in the cultured dental follicle cells is enhanced by each of the four putative tooth eruption molecules examined. This is confirmed in vivo in that injections of either EGF or IL-1a will enhance MCP-1 expression (Figs. 1 and 10). All of the four molecules are transcribed and translated either in the dental follicle or the adjacent stellate reticulum—EGF (Wise et al., 1992; Lin et al., 1996), TGF-b1 (Wise and Fan, 1991; Lin and Wise, 1993), IL-1a (Wise et al., 1994, 1995b) and CSF-1 (Wise and Lin, 1994; Wise et al., 1995b). All also achieve their peak transcription and translation early postnatally. Thus, the follicle is receptive to a multitude of signals to enhance MCP-1 transcription for the ultimate production of MCP-1 for monocyte recruitment. The optimal concentrations of the mediators of MCP-1 expression are physiological meaningful, at least when compared with their concentrations in other tissues and serum. Their concentration in the dental follicle and stellate reticulum is unknown except for the observation that their maximal gene expression and immunostaining occur early postnatally (see review by Wise and Lin, 1995). Regarding TGF-b1, the 2-ng/ml concentration for maximal MCP-1 expression would be less than the 8-ng TGF-b1/mg protein in rat bone extracts (Carrington et al., 1988) or the 430-ng TGF-b1/g (wet weight) found in platelets (Assoian et al., 1983). The optimal concentration of CSF-1 used (40 ng/ml) is the same concentration present in fetal mouse serum (Roth and Stanley, 1996). For a tissue, CSF-1 is present in the mouse kidney at a concentration of 70 ng/mg protein (Roth and Stanley, 1996). EGF and IL-1a were injected at a concentration of 1 µg/g body weight and in vitro were optimal mediators at 25–50 ng/ml. As detailed by Carpenter and Wahl (1991), EGF has a wide range of concentration in body fluids that includes a low of 1.14 ng/ml serum to such highs as 101 ng/ml of seminal fluid, 140 ng/ml milk and 272 ng/ml prostate fluid. IL-1a concentration reports are scarce but extraction of IL-1a from brown adipocytes or spleen shows a high concentration, e.g., 260–460 ng/mg protein in the nuclear fraction alone (Burysek and MCP-1 EXPRESSION IN THE DENTAL FOLLICLE Houstek, 1996). It also should be noted that studies using articular cartilage slices used a concentration of 50 ng IL-1a/ml medium for 4 weeks (Xu et al., 1996). These molecules that enhance MCP-1 gene expression probably have other roles in tooth eruption, as shown by their ability to enhance gene expression in a manner such that a cascade of molecular signals (EGF=TGF-b1=IL-1a=CSF-1) might initiate eruption (Wise and Lin, 1995). Although CSF-1 might be the critical molecule needed to enhance MCP-1 gene expression, the fact that the other putative eruption molecules also can increase MCP-1 expression reflects the theme that more than one molecule can exert the same developmental effect. This redundancy of gene function is demonstrated by the example that either transforming growth factor-alpha (TGF-a) or epidermal growth factor (EGF) can accelerate incisor eruption (Tam, 1985; Cohen, 1962). Mice with a null mutation of the TGF-a gene still have a normal eruption of the incisors (Mann et al., 1993), suggesting that EGF substitutes for the missing TGF-a. MCP-1 itself is a CC chemokine, so named because of two conserved cysteine residues that are adjacent to each other near the N-terminal end of the chemotactic protein (see review by Rollins, 1997). Human monocyte chemotactic protein 1 and its murine counterpart (JE) have a 62% amino acid homology over the first 68 positions of the protein (Rollins et al., 1989) and both are equally chemoattractive for monocytes (Ernst et al., 1994). In view of their structural and functional similarities, it is probable that the role MCP-1 could play in molar eruption in the rat is paralleled in the human. Tooth eruption is somewhat analogous to chronic inflammation in that both processes involve the attraction of monocytes to a specific site by a chemotactic substance. For example, expression of MCP-1 is noted in osseous inflammation (Rahimi et al., 1995), inflammation of gingiva (Yu and Graves, 1995), atherosclerosis (Nelken et al., 1991; Yla-Herttuala et al., 1991; Yu et al., 1992), synovial inflammation (Villiger et al., 1992) and glomerulonephritis (Rovin et al., 1994). As would be expected, MCP-1 expression is often enhanced by proinflammatory cytokines such as IL-1 (Brieland et al., 1995; Goppelt-Struebe and Stroebel, 1995; Rovin et al., 1992; Takeshita et al., 1993), TGF-b1 (Hanazawa et al., 1991; Takeshita et al., 1995) and CSF-1 (Shyy et al., 1993). The probability that many of these proinflammatory cytokines may be involved in tooth eruption suggests that a developmental process, tooth eruption, may have evolved as a modification of the chronic inflammation process. In fact, MCP-1 itself stimulates histamine release from basophils (Alam et al., 1992; Bischoff et al., 1992; Kuna et al., 1992), again relating tooth eruption to an inflammation heritage. EXPERIMENTAL PROCEDURES Cell Cultures Harlan Sprague-Dawley rats were housed in Association for Accreditation and Assessment of Laboratory 349 Animal Care (AAALAC)-approved facilities and provided with water and food ad libitum. Cultures of dental follicle cells were established from the first and second mandibular molars obtained from 6–7-day-old postnatal rats (Wise et al., 1992). The cells were maintained in Eagle’s modified minimal essential medium (MEM) (ICN Biochemicals, Costa Mesa, CA) containing 15% fetal bovine serum (Gibco-BRL, Grand Island, NY) in the presence of streptomycin and penicillin. For the experiments in which cells were cultured with different cytokines or growth factors, the fourth passage or later of the dental follicle cells was used to insure the homogeneity of the cell population (Wise et al., 1992). Incubation and Injection Experiments Dental follicle cells were incubated separately with TGF-b1 (R & D Systems, Minneapolis, MN), EGF (Upstate Biotechnology, Lake Placid, NY), IL-1a (gift from NCI Biological Resources Branch, Frederick, MD), and murine CSF-1 (R & D Systems) to determine both concentration and time-dependent effects of these agents on MCP-1 gene expression. In terms of concentration effects, dosages of TGF-b1 ranged from 0 (control) to 4 ng/ml medium; EGF from 0 to 250 ng/ml; IL-1a from 0 to 250 ng/ml; and CSF-1 from 0 to 100 ng/ml. For all the dosage studies, the cells were incubated for 3 hr for any given concentration. For the time-course studies, incubation times of TGF-b1 ranged from 0 (control) to 24 hr; EGF ranged from 0 to 24 hr; IL-1a from 0 to 30 hr; and CSF-1 from 0 to 24 hr. For all the above experiments, the dental follicle cells first were cultured in MEM/15% fetal bovine serum until near confluency at 37°C and 5% CO2 in air. Next, the cells were washed with serum-free MEM, incubated for 3 hr and then treated for the times or dosages listed above in the presence of serum-free MEM. For the IL-1a injection experiments, rats were injected with IL-1a at a concentration of 1 µg IL-1a/g body weight on days 2, 6 or 9. Controls were injected with an equal volume of saline. Fifteen hours after injections (days 3, 7 or 10), the animals were sacrificed and the dental follicles removed from the first mandibular molars. The total RNA was isolated from the experimentals and controls according to the TRI REAGENT protocol (Molecular Research Center, Cincinnati, OH). These experiments were repeated three times and for each age (days 2, 6 or 9) six rats were injected— three experimentals and three controls. For the EGF injection experiments, 12 rats were injected daily (starting at day 0 postnatally) with EGF at a concentration of 1.0 µg/g body weight. For controls, another 12 rats of the same age were injected daily with the same volume of saline. At days 1, 3, 5 and 7, at 2–3 hr after the injection on that given day, three experimentals and three controls were sacrificed. The total RNA was then isolated from the dental follicles for reverse transcription-polymerase chain reaction (RT-PCR) analysis. These injection experiments were repeated twice in their entirety. 350 QUE AND WISE Reverse Transcription-Polymerase Chain Reaction After isolation of the total RNA, it was quantitated at an optical reading of 260 nm and the ratio OD 260/280 was greater than 1.6. The total RNA was reverse transcribed as previously described (Que and Wise, 1997). The specific primers for the PCR were chosen based on the nucleotide sequence of MCP-1 cDNA of the rat (Yoshimura et al., 1991). The 58 primer sequence was ATGCAGGTCTCTGTCACGCTTCTG and the 38 primer sequence was TCCCATTCATCTCTATACAT. Details of the PCR technique used have been published (Que and Wise, 1997). In general, 2 µL of the reverse transcription product was mixed with the primers, Taq polymerase, nucleotides and PCR buffer to make a total volume of 25 µL. For controls, primers for the b-actin gene (Clontech) were used in parallel amplification with the target gene (MCP-1). The reaction was conducted using a Perkin Elmer Thermal Cycler programmed at 95°C for 1 min (denaturation), 60°C for 1 min (annealing) and 72°C for 2 min (extension) for 30 cycles. Following PCR, 5 µL of the entire reaction volume was electrophoresed on a 1.1% agarose gel. Each gel was then stained with ethidium bromide and photographed. The RT-PCR methods of detection used were within the linear range for detection of RNA. Reverse transcription of total RNA ranging from 1 to 5 µg demonstrated a linear increase in the PCR product (data not shown). Linearity also was shown for the PCR cycles in the range of 20–40 cycles. ACKNOWLEDGMENTS This work was supported by National Institute of Dental Research grant DE08911 to G.E. Wise. The authors thank Ms. Cindy Daigle for the typing of this manuscript and for assistance in preparing the figures. We also are indebted to Mr. Charles Adams and Ms. Angela Layfield for their technical assistance. 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