Journal of Cellular Physiology

Mizuguchi, Toru; Mitaka, Toshihiro; Hirata, Koichi; Oda, Hiroaki; Mochizuki, Yohichi

doi: 10.1002/(SICI)1097-4652(199803)174:3<273::AID-JCP1>3.0.CO;2-Lpmid: 9462689

In the present study, we showed the role of the liver‐enriched transcription factors in the transition during which proliferating hepatocytes become quiescent. We used primary rat hepatocytes cultured in modified L‐15 medium. The cells proliferated and, after the addition of 2% dimethyl sulfoxide (DMSO) from day 4, they stopped growing and gradually differentiated. During hepatic proliferation, expression of hepatocyte nuclear factors (HNF)1α, HNF4, C/EBPα, and C/EBPβ mRNAs was depressed, whereas that of HNF3α and HNF3β transcripts was enhanced. After the addition of DMSO, the expression of HNF1α, HNF3γ, and HNF4 returned to the level in isolated cells and HNF1β mRNA expression gradually increased. However, expression of C/EBPα and C/EBPβ mRNAs was partially recovered. The mitoinhibitory agents, IL‐1β, IL‐6, TGF‐β, and activin A, were examined to determine whether they could induce differentiation of proliferating hepatocytes as shown in cells treated with DMSO. Although these factors inhibited cell growth, the cells did not differentiate. The expression pattern of HNF3γ mRNA was quite different in the cells cultured with DMSO and those cultured with cytokines. Therefore, hepatic differentiation requires not only inhibition of DNA synthesis but also induction of appropriate transcription factors. Thus, expression of HNF3γ, C/EBPα, and C/EBPβ may be necessary for hepatocytes to acquire highly differentiated functions in addition to coexpression of certain amounts of transcripts of HNF1α, HNF1β, HNF3α, HNF3β, and HNF4 as well as suppression of C/EBPδ. J. Cell. Physiol. 174:273–284, 1998. © 1998 Wiley‐Liss, Inc.

Fine, Alan; Miranda, Kathleen; Farmer, Stephen R.; Anderson, Nichole L.

doi: 10.1002/(SICI)1097-4652(199803)174:3<285::AID-JCP2>3.0.CO;2-Kpmid: 9462690

Fas, which functions to initiate a signal causing apoptosis, is expressed in epithelia, thus, suggesting a role in controlling cell number during states of cell and matrix turnover. In view of this, we hypothesized that cell‐matrix interactions may be an important determinant of Fas expression in epithelial cells. To investigate this, we examined the effect of insoluble extracellular matrix molecules on Fas expression in murine lung epithelial (MLE) cells, a transformed mouse lung epithelial cell line. We report that (1) insoluble extracellular matrices increased Fas mRNA in a time and concentration‐dependent manner; (2) induced increases in Fas mRNA were associated with concomitantly increased Fas protein; and (3) nonspecific adherence to a polylysine substrate did not induce Fas mRNA. Consistent with these findings, Fas‐induced apoptosis was significantly enhanced in cultures plated on type IV collagen. Employing rat hepatocytes, we confirmed that the insoluble extracellular matrix also increases Fas expression in primary epithelial cells. By amplifying Fas‐mediated apoptosis, these data suggest a mechanism whereby the extracellular matrix regulates the fate of specific epithelial cell populations. J. Cell. Physiol. 174:285–292, 1998. © 1998 Wiley‐Liss, Inc.

McCusker, Robert H.; Clemmons, David R.

doi: 10.1002/(SICI)1097-4652(199803)174:3<293::AID-JCP3>3.0.CO;2-Jpmid: 9462691

The modulation of insulin‐like growth factor‐binding protein (IGFBP) secretion is an important variable affecting muscle cell metabolism, proliferation, and differentiation. We have previously shown that secretion of IGFBP‐4 and IGFBP‐5 by L6 and BC3H‐1 muscle cells was stimulated by treatment with either insulin, IGF‐I, or IGF‐II. Herein, these cells were used to further identify mechanisms involved in controlling IGFBP secretion. Agents that elevate intracellular cAMP concentrations (dcAMP, forskolin, isoproterenol, and prostaglandin [PGE1]) increase secretion of IGFBP‐4 and IGFBP‐5 from L6 cells. Similar increases in IGFBP secretion were found by treatment with either insulin, IGF‐I, or dcAMP. The effects of dcAMP and either insulin or IGF‐I were additive, but the effects of insulin and IGF‐I were not additive. These results suggest that insulin/IGF‐I and dcAMP are acting via distinct mechanisms to stimulate IGFBP secretion. Indomethacin, which blocks endogenous prostaglandin synthesis, and progesterone, which decreases intracellular cAMP levels, decreased IGFBP‐4 and IGFBP‐5 secretion. IGFBP‐5 secretion by BC3H‐1 cells was increased by either insulin or IGF‐I. Agents which elevate intracellular cAMP concentrations did not increase IGFBP‐5 secretion. Additionally, these agents were not synergistic with either insulin or IGF‐I. However, indomethacin and progesterone depressed IGFBP‐5 secretion by BC3H‐1 cells. In summary, there appear to be at least two intracellular signaling mechanisms controlling IGFBP‐4 and IGFBP‐5 secretion by L6 and BC3H‐1 muscle cells. IGFBP secretion by L6 cells is stimulated by both insulin/IGF‐I and cAMP‐dependent pathways, whereas IGFBP‐5 secretion by BC3H‐1 cells is stimulated only by the insulin/IGF pathway. IGFBP secretion by both cell lines can be decreased by agents which depress cAMP levels. Our results suggest that two divergent but synergistic pathways modulate IGFBP production and these mechanisms can potentially modulate IGF activity during muscle cell proliferation and differentiation. J. Cell. Physiol. 174:293–300, 1998. © 1998 Wiley‐Liss, Inc.

Ghahary, Aziz; Shen, Qiong; Shen, You J.; Scott, Paul G.; Tredget, Edward E.

doi: 10.1002/(SICI)1097-4652(199803)174:3<301::AID-JCP4>3.0.CO;2-Spmid: 9462692

Transforming growth factor β1 (TGF‐β1) belongs to a family of multifunctional modulatory proteins involved in cell growth, differentiation, development, and wound healing. Although the biological activities of TGF‐β1 have been extensively studied, its regulation remains obscure. Here we report the effects of insulin‐like growth factor‐1 (IGF‐1) on the expression of TGF‐β1 by dermal fibroblasts and suggest a possible mechanism. An enzyme‐linked immunosorbent assay (ELISA) specific for TGF‐β revealed a greater than twofold increase (12.3 ± 1.6 vs. 4.8± 0.8 pg/104 cells, n = 7, P < 0.05) in the protein in conditioned medium obtained from IGF‐1‐treated cells compared to that from untreated controls. Similar results were obtained by the mink lung epithelial cell growth inhibition assay. The results of Northern analysis revealed a dose‐dependent increase in TGF‐β1 mRNA in response to IGF‐1 treatment. Using the optimum concentration of IGF‐1 (100 ng/ml), a greater than twofold increase (25.43 ± 5.7 vs. 12.13 ± 4.5, P < 0.05) in TGF‐β1 mRNA was observed. This effect persisted for at least 48 h after IGF‐1 was removed from the culture medium. Nuclear run‐on assay showed that this stimulation was due, at least in part, to an increase in the rate of transcription of the TGF‐β1 gene. Treatment of human dermal fibroblasts with IGF‐1 caused a substantial increase in c‐fos and c‐jun mRNA expression within 30 and 60 min, respectively. In contrast to c‐jun mRNA which was constitutively expressed by dermal fibroblasts, the expression of c‐fos mRNA was transient and only detectable between 15 and 60 min. Greater than 58% of the increase in TGF‐β1 caused by IGF‐1 could be blocked by the addition of anti‐TGF‐β1 neutralizing antibody to the culture medium, suggesting that autoinduction of TGF‐β1 may be involved. An increase in IGF‐1‐induced TGF‐β1 should be important in many different physiological processes such as cellular proliferation, differentiation, and wound healing. These findings also suggest that induction of TGF‐β1 mRNA and protein by IGF‐1 may be a mechanism by which this cytokine is regulated in physiological and/or pathological conditions. J. Cell. Physiol. 174:301–309, 1998. © 1998 Wiley‐Liss, Inc.

Filla, Mark S.; Dam, Phoungan; Rapraeger, Alan C.

doi: 10.1002/(SICI)1097-4652(199803)174:3<310::AID-JCP5>3.0.CO;2-Rpmid: 9462693

Binding of fibroblast growth factors (FGFs) to receptor tyrosine kinases (FGFRs) and signaling is facilitated by binding of FGF to heparan sulfate proteoglycans (HSPGs). There are multiple families of HSPGs, including extracellular and cell surface forms. An important and potentially controversial question is whether cell surface forms of HSPGs act as positive or negative regulators of FGF signaling. This study examines the ability of the cell surface HSPG syndecan‐1 to regulate FGF binding and signaling. HSPG‐deficient Raji lymphoma cells, expressing a transfected syndecan‐1 cDNA (Raji S1 cells), were used as HSPG “donor” cells. BaF3 cells, expressing an FGFR1 cDNA (FR1C‐11 cells), were used as FGFR “reporter” cells. Using Raji S1 cells preincubated with FGF, it was found that they formed heterotypic aggregates with FR1C‐11 cells in the presence of FGF‐2, but not FGF‐1. In addition, the FR1C‐11 cells demonstrated FGF‐2, but not FGF‐1, dependent survival when cultured on fixed Raji S1 cells. Thus, Raji syndecan‐1 (1) differentially regulates the binding and signaling of FGFs 1 and 2 and (2) acts as a positive regulator of FGF‐2 signaling. J. Cell. Physiol. 174:310–321, 1998. © 1998 Wiley‐Liss, Inc.

Mendoza, Lorea; Olaso, Elvira; Anasagasti, Miren J.; Fuentes, Angela M.; Vidal‐Vanaclocha, Fernando

doi: 10.1002/(SICI)1097-4652(199803)174:3<322::AID-JCP6>3.0.CO;2-Qpmid: 9462694

The role of mannose receptors from hepatic sinusoidal endothelium (HSE) in liver colonization by B16 melanoma (B16M) cells was studied. The expression of high mannose‐type oligosaccharides on the surface of B16M cells was enhanced by in vitro treatment with 1‐deoximannojirimycin (1‐DMM). There was a significant (P < 0.01) enhancement of hepatic metastasis when B16M cells were 1‐DMM‐treated before being intrasplenically injected into C57BL/6J mice. Intraperitoneal administration of 5 mg/kg recombinant human interleukin‐1 receptor antagonist (rHuIL‐1Ra) inhibited the 1‐DMM‐induced enhancement of metastasis. Expression of high mannose‐type oligosaccharides on the surface of 1‐DMM‐treated B16M cells and their in vitro adhesion to the HSE was significantly correlated (R = 0.82). The addition of either 100 μg/ml mannan or paraformaldehyde (PFA)‐fixed 1‐DMM‐treated B16M cells to cultured HSE for a period of 12 h significantly (P < 0.01) increased the release of IL‐1β from the HSE compared to that liberated by the HSE incubated with either basal medium or PFA‐fixed untreated B16M cells. The same HSE treatments also significantly (P < 0.01) increased the degree of adhesion of other B16M cells to HSE, being abrogated by anti‐mouse vascular cell adhesion molecule‐1 (VCAM‐1) antibodies. The conditioned media from HSE cultures, activated by PFA‐fixed, 1‐DMM‐treated B16M cells significantly (P < 0.01) increased B16M cell proliferation when compared to conditioned media from HSE cultures incubated with PFA‐fixed, untreated B16M cells. Thus, 1‐DMM treatment of B16M cells enhanced the development of hepatic metastasis by IL‐1‐dependent mechanisms. The mechanism is consistent with in vitro mannose receptor‐mediated melanoma cell attachment to the HSE, which subsequently upregulates IL‐1β release, VCAM‐1‐dependent adherence, and melanoma growth factor(s) release by HSE. J. Cell. Physiol. 174:322–330, 1998. © 1998 Wiley‐Liss, Inc.

Venezian, Rachel; Shenker, Bruce J.; Datar, Sugandha; Leboy, Phoebe S.

doi: 10.1002/(SICI)1097-4652(199803)174:3<331::AID-JCP7>3.0.CO;2-Ppmid: 9462695

Chondrocytes show an unusual ability to thrive under serum‐free conditions as long as insulin, thyroxine, and cysteine are present. Studies with sternal chondrocytes from chick embryos indicate that thymidine incorporation in chondrocytes cultured under serum‐free conditions is 30–50% of that seen with fetal bovine serum (FBS). In contrast, skin fibroblast proliferation in serum‐free culture is <5% of that seen with serum. Addition of 30–50 μM ascorbic acid to serum‐free medium stimulates chondrocyte proliferation 4–5×, resulting in levels of thymidine incorporation higher than that seen with 10% serum. Three to five hours of ascorbate exposure is sufficient to stimulate proliferation, with maximal stimulation seen after 12–15 h. Bromo‐deoxyuridine (BrdU) labelling indicated that approximately 25% of chondrocytes transit S phase during a 4‐h period (16–20 h after ascorbate). Once maximal stimulation is reached, the proliferation rate remains fairly constant over at least 40 h. Ascorbate therefore increases the steady‐state level of chondrocytes in the cycle. Because the stimulation of chondrocyte proliferation was greater than the net increase in cell numbers, we examined the level of apoptosis. Nuclear morphology, terminal uridine nucleotide end‐labelling (TUNEL) assay, and 7‐AAD/Hoechst dye FACS analyses all indicated that approximately 15% of the ascorbate‐treated chondrocytes were undergoing apoptosis, while only 5% of the control chondrocytes were apoptotic. When prehypertrophic chondrocytes from the cephalic region of embryonic sternae were stimulated to undergo hypertrophy with rhBMP‐2 + ascorbate, levels of apoptosis were similar to that seen with ascorbate alone. In contrast, treatment of caudal chondrocytes with BMP plus ascorbate does not induce hypertrophy, and the proportion of apoptotic cells was less than that seen with ascorbate alone. These results imply that in chondrocytes the transition to hypertrophy is associated with a decreased number of proliferating cells and a relatively high level of apoptosis. J. Cell. Physiol. 174:331–341, 1998. © 1998 Wiley‐Liss, Inc.

Aimes, Ronald T.; Li, Ling‐Hui; Weaver, Brian; Hawkes, Susan; Hahn‐Dantona, Elizabeth A.; Quigley, James P.

doi: 10.1002/(SICI)1097-4652(199803)174:3<342::AID-JCP8>3.0.CO;2-Opmid: 9462696

Rous sarcoma virus‐transformed chicken embryo fibroblasts (RSVCEF), when compared to normal CEF, produce elevated levels of matrix metalloproteinase‐2 (MMP‐2) that exists in a form free of complexed tissue inhibitor of metalloproteinase‐2 (TIMP‐2). In order to ascertain whether the increased levels of TIMP‐free MMP‐2 in RSVCEF cultures are due to diminished expression of TIMP‐2 or alterations in TIMP‐2 that diminish its MMP‐2 binding ability, it was necessary to clone, characterize, and express chicken TIMP‐2 cDNA. The TIMP‐2 cDNA was cloned from a chick embryo λgt11 library by RT‐PCR using primers based on amino‐acid sequences determined from isolated TIMP‐2. The deduced amino acid sequence for chicken TIMP‐2 is 81% identical to human TIMP‐2; most of the sequence differences lie in the carboxyl terminal portion of chicken TIMP‐2. Northern analysis of mRNA levels in CEF and RSVCEF demonstrates that TIMP‐2 mRNA levels are increased in RSVCEF. However, TIMP‐2 protein levels, relative to proMMP‐2 levels, appear to decrease upon transformation and suggest additional control of TIMP‐2 at the post‐transcriptional level. Addition of recombinantly expressed TIMP‐2 to RSVCEF cultures causes a disappearance of TIMP‐free (TF) proMMP‐2 with a corresponding increase in the TIMP‐complexed (TC) proMMP‐2 levels, demonstrating that TF proMMP‐2 is capable of converting to TC pro‐MMP‐2 when free TIMP‐2 is available. Surprisingly, RSVCEF cultures manifest a TIMP‐2 population that is not complexed to MMP‐2, despite the coexistence of TIMP‐free proMMP‐2. Gel‐filtration analysis indicates that this uncomplexed TIMP‐2 exhibits an apparent molecular weight of 50 kDa, indicating it is not free TIMP‐2 and that it exists in transformed cultures in a noncovalent complex with an undefined molecule. Thus transformed cells can alter the TIMP‐2/MMP‐2 balance by transcriptional and post‐translational modifications, yielding a population of inhibitor‐free, proteolytically active MMP2. J. Cell. Physiol. 174:342–352, 1998. © 1998 Wiley‐Liss, Inc.

Haase, Helen R.; Clarkson, Richard W.; Waters, Michael J.; Bartold, P. Mark

doi: 10.1002/(SICI)1097-4652(199803)174:3<353::AID-JCP9>3.0.CO;2-Npmid: 9462697

In order to understand the relationship between specific growth factors and matrix synthesis by periodontal cells, we have investigated the effects of platelet‐derived growth factor BB (PDGF‐BB), insulin‐like growth factor‐I (IGF‐1), and growth hormone on DNA and proteoglycan synthesis by cultured human gingival and periodontal ligament fibroblasts in vitro. PDGF‐BB and IGF‐1, but not growth hormone, were mitogenic for both periodontal ligament fibroblasts and gingival fibroblasts, although the periodontal ligament cells responded more strongly. The mitogenic response was accompanied by alterations in expression of matrix proteoglycan mRNA. For both the gingival and periodontal ligament cells, there was a decrease in mRNA for decorin and an increase in mRNA for versican following exposure to IGF‐1 and PDGF‐BB. Although no change was seen in response to PDGF, biglycan mRNA level was increased by IGF‐1 in periodontal ligament fibroblasts. With the gingival fibroblats, biglycan mRNA levels were unaffected by IGF‐1, PDGF‐BB, or growth hormone. These findings suggest variable responses of fibroblasts to growth factors depending upon anatomical site within the periodontium. Moreover, there appears to be a correlation between cell proliferation and the types of proteoglycan synthesised with decorin expression being suppressed, and versican being increased during fibroblast proliferation. J. Cell. Physiol. 174:353–361, 1998. © 1998 Wiley‐Liss, Inc.

Engle, M. J.; Goetz, G. S.; Alpers, D. H.

doi: 10.1002/(SICI)1097-4652(199803)174:3<362::AID-JCP10>3.0.CO;2-Bpmid: 9462698

Caco‐2 cells are derived from a human colonic adenocarcinoma, but differentiate into small intestinal‐like cells after confluence. While this enterocytic differentiation has been well studied, the presumed parallel loss of colonocyte function has not been as thoroughly examined. To follow the phenotype for both tissues, Western blots were performed using antisera recognizing liver/bone/kidney alkaline phosphatase and surfactant‐like particle proteins found in normal human colon, along with antisera against the small bowel representatives of the same proteins. Antisera against proteins enriched in either enterocytes (α1‐antitrypsin) or colonocytes (surfactant protein A) were also evaluated. Alkaline phosphatase activity increased from 3 to 18 days post‐confluence. Activity at 3 days post‐confluence derived substantially from both isomers. Thereafter, the colonic (liver/bone/kidney) isomer declined to low levels as the content of the enterocytic isomer rose. A similar pattern was found with colonic (decreasing expression) and enterocytic (increasing expression) surfactant‐like particle proteins. In particular, the content of larger enterocytic particle proteins (97 and 116 kDa) increased with time in culture. Expression of α1‐antitrypsin increased early and remained high, whereas surfactant protein A generally declined after the third day post‐confluency. In summary, undifferentiated Caco‐2 cells express very low levels of proteins characteristic of either colonocytes or enterocytes. Immediately after confluence, they expressed proteins characteristic of both cell types. Thereafter, the content of colonocyte‐specific proteins decreased, whereas those specific for the enterocyte increased. The timing and degree of this phenotypic switch have implications for the interpretation of experiments using Caco‐2 cells as a model of small intestinal function. J. Cell. Physiol. 174:362–369, 1998. © 1998 Wiley‐Liss, Inc.