Identification and Contribution of Inflammation-Induced Novel MicroRNA in the Pathogenesis of Systemic Lupus Erythematosus.

Recently microRNAs (miRNAs) have been recognized as powerful regulators of many genes and pathways involved in the pathogenesis of inflammatory diseases including Systemic Lupus Erythematosus (SLE). SLE is an autoimmune disease characterized by production of various autoantibodies, inflammatory immune cells, and dysregulation of epigenetic changes. Several candidate miRNAs regulating inflammation and autoimmunity in SLE are described. In this study, we found significant increases in the expression of miR21, miR25, and miR186 in peripheral blood mononuclear cells (PBMCs) of SLE patients compared to healthy controls. However, miR146a was significantly decreased in SLE patients compared to healthy controls and was negatively correlated with plasma estradiol levels and with SLE disease activity scores (SLEDAI). We also found that protein levels of IL-12 and IL-21 were significantly increased in SLE patients as compared to healthy controls. Further, our data shows that protein levels of IL-12 were positively correlated with miR21 expression and protein levels of IL-21 positively correlated with miR25 and miR186 expression in SLE patients. In addition, we found that levels of miR21, miR25, and miR186 positively correlated with SLEDAI and miR146a was negatively correlated in SLE patients. Thus, our data shows a dynamic interplay between disease pathogenesis and miRNA expression. This study has translational potential and may identify novel therapeutic targets in patients with SLE.

Cellular and Molecular Phenotypes of pConsensus Peptide (pCons) Induced CD8+ and CD4+ Regulatory T Cells in Lupus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with widespread inflammation, immune dysregulation, and is associated with the generation of destructive anti-DNA autoantibodies. We have shown previously the immune modulatory properties of pCons peptide in the induction of both CD4+ and CD8+ regulatory T cells which can in turn suppress development of the autoimmune disease in (NZB/NZW) F1 (BWF1) mice, an established model of lupus. In the present study, we add novel protein information and further demonstrate the molecular and cellular phenotypes of pCons-induced CD4+ and CD8+ Treg subsets. Flow cytometry analyses revealed that pCons induced CD8+ Treg cells with the following cell surface molecules: CD25highCD28high and low subsets (shown earlier), CD62Lhigh, CD122low, PD1low, CTLA4low, CCR7low and 41BBhigh. Quantitative real-time PCR (qRT-PCR) gene expression analyses revealed that pCons-induced CD8+ Treg cells downregulated the following several genes: Regulator of G protein signaling (RGS2), RGS16, RGS17, BAX, GPT2, PDE3b, GADD45ß and programmed cell death 1 (PD1). Further, we confirmed the down regulation of these genes by Western blot analyses at the protein level. To our translational significance, we showed herein that pCons significantly increased the percentage of CD8+FoxP3+ T cells and further increased the mean fluorescence intensity (MFI) of FoxP3 when healthy peripheral blood mononuclear cells (PBMCs) are treated with pCons (10 µg/ml, for 24-48 hours). In addition, we found that pCons reduced apoptosis in CD4+ and CD8+ T cells and B220+ B cells of BWF1 lupus mice. These data suggest that pCons stimulates cellular, immunological, and molecular changes in regulatory T cells which in turn protect against SLE autoimmunity.

Interferon Genes Are Influenced by 17ß-Estradiol in SLE.

Recent evidence suggests the existence of a nexus between inflammatory pathways and the female sex hormone 17ß-estradiol, resulting in increased interferon-stimulated genes (ISGs), autoantibodies, and dysregulation of immune cells in SLE. However, the molecular mechanisms and the effect of estradiol on candidate target genes and their pathways remains poorly understood. Our previous work suggests that female SLE patients have increased estradiol levels compared to healthy controls. In the present study, we explored the effects of 17ß-estradiol treatment on expression of IFN (interferons)-stimulated genes and pro-inflammatory cytokines/chemokines. We found significantly increased (5-10-fold) expression of IFN-regulated genes in healthy females. Furthermore, we found significantly increased plasma levels of IL-6, IL-12, IL-17, IL-18, stem cell factor (SCF), and IL-21/IL-23 in SLE patients compared to healthy controls, and those levels positively correlated with the plasma levels of 17ß-estradiol. In addition, levels of IL-21 positively correlated with the SLE disease activity index (SLEDAI) score of SLE patients. In vitro treatment of PBMCs from either SLE patients or healthy controls with 17ß-estradiol at physiological concentration (~50 pg/ml) also significantly increased secretion of many pro-inflammatory cytokines and chemokines (IL-6, IL-12, IL-17, IL-8, IFN-γ; MIP1α, and MIP1ß) in both groups. Further our data revealed that 17ß-estradiol significantly increased the percentage of CD3+CD69+ and CD3+IFNγ+ T cells; whereas, simultaneous addition of 17ß-estradiol and an ERα inhibitor prevented this effect. Collectively, our findings indicate that 17ß-estradiol participates in the induction of pro-inflammatory cytokines and chemokines and further influences interferon genes and pathways.

Effects of Peptide-Induced Immune Tolerance on Murine Lupus.

The regulation of autoimmunity and the molecular mechanisms by which different immune cells, including T cells, polymorphonuclear leukocytes (PMN-granulocytes), and B cells suppress autoimmune diseases is complex. We have shown previously that BWF1 lupus mice are protected from autoimmunity after i.v. injection or oral administration of tolerogenic doses of pCons, an artificial synthetic peptide based on sequences containing MHC class I and MHC class II determinants in the VH region of a J558-encoded BWF1 anti-DNA Ab. Several T cell subsets can transfer this tolerance. In this study, we determined the potential roles of granulocytes, B cells and regulatory T cells altered by pCons treatment in the BWF1 (NZB/NZW) mouse model of lupus. Immunophenotyping studies indicated that pCons treatment of BWF1 mice significantly increased CD4+FoxP3+ T cells, reduced the percent of B cells expressing CD19+CD5+ but increased the percent of CD19+CD1d+ regulatory B cells and increased the ability of the whole B cell population to suppress IgG anti-DNA production in vitro. pCons treatment significantly decreased the expression of CTLA-4 (cytotoxic T-lymphocyte-associated protein-4) in CD8+ T cells. In addition, peptide administration modified granulocytes so they became suppressive. We co-cultured sorted naïve B cells from mice making anti-DNA Ab (supported by addition of sorted naive CD4+ and CD8+ T cells from young auto-antibody-negative BWF1 mice) with sorted B cells or granulocytes from tolerized mice. Both tolerized granulocytes and tolerized B cells significantly suppressed the production of anti-DNA in vitro. In granulocytes from tolerized mice compared to saline-treated littermate controls, real-time PCR analysis indicated that expression of interferon-induced TNFAIP2 increased more than 2-fold while Ptdss2 and GATA1 mRNA were up-regulated more than 10-fold. In contrast, expression of these genes was significantly down-regulated in tolerized B cells. Further, another IFN-induced protein, Bcl2, was reduced in tolerized B cells as determined by Western blot analyses. In contrast, expression of FoxP3 was significantly increased in tolerized B cells. Together, these data suggest that B cells and granulocytes are altered toward suppressive functions by in vivo tolerization of BWF1 mice with pCons and it is possible these cell types participate in the clinical benefits seen in vivo.

Sex Hormones and Gender Influence the Expression of Markers of Regulatory T Cells in SLE Patients.

Regulatory T cells have been implicated in the regulation and maintenance of immune homeostasis. Whether gender and sex hormones differentially influence the expression and function of regulatory T cell phenotype and their influence on FoxP3 expression remains obscure. We provide evidence in this study that the number and percent of human regulatory T cells (Tregs) expressing CD4+ and CD8+ are significantly reduced in healthy females compared to healthy males. In addition, both CD4+CD25+hi and CD8+CD25+hi subsets in healthy males have a 2-3 fold increase in FoxP3 mRNA expression compared to healthy females. Female SLE patients, compared to healthy women, have elevated plasma levels of estradiol and decreased levels of testosterone. Higher levels of testosterone correlate with higher expression of FoxP3 in CD4+CD25hiCD127low putative Tregs in women with SLE. Incubation of CD4+ regulatory T cells with 17ß-estradiol at physiological levels generally decreased FoxP3 expression in females with SLE. These data suggest that females may be more susceptible than males to SLE and other autoimmune diseases in part because they have fewer Tregs and reduced FoxP3 expression within those cells due to normal E2 levels which suppress FoxP3 expression. In addition, low levels of plasma testosterone in women may further reduce the ability of the Tregs to express FoxP3. These data suggest that gender and sex hormones can influence susceptibility to SLE via effects on regulatory T cells and FoxP3 expression.

CD8+ T regulatory cells in lupus.

T regulatory cells (Tregs) have a key role in the maintenance of immune homeostasis and the regulation of immune tolerance by preventing the inflammation and suppressing the autoimmune responses. Numerical and functional deficits of these cells have been reported in systemic lupus erythematosus (SLE) patients and mouse models of SLE, where their imbalance and dysregulated activities have been reported to significantly influence the disease pathogenesis, progression and outcomes. Most studies in SLE have focused on CD4+ Tregs and it has become clear that a critical role in the control of immune tolerance after the breakdown of self-tolerance is provided by CD8+ Tregs. Here we review the role, cellular and molecular phenotypes, and mechanisms of action of CD8+ Tregs in SLE, including ways to induce these cells for immunotherapeutic modulation in SLE.

Metabolic relationship between diabetes and Alzheimer's Disease affected by Cyclo(His-Pro) plus zinc treatment.

BACKGROUND: Association of Alzheimer's Disease (AD) with Type 2 Diabetes (T2D) has been well established. Cyclo(His-Pro) plus zinc (Cyclo-Z) treatment ameliorated diabetes in rats and similar improvements have been seen in human patients. Treatment of amyloid precursor protein (APP) transgenic mice with Cyclo-Z exhibited memory improvements and significantly reduced Aß-40 and Aß-42 protein levels in the brain tissues of the mice. SCOPE OF REVIEW: Metabolic relationship between AD and T2D will be described with particular attention to insulin sensitivity and Aß degradation in brain and plasma tissues. Mechanistic effect of insulin degrading enzyme (IDE) in decreasing blood glucose and brain Aß levels will be elucidated. Cyclo-Z effects on these biochemical parameters will be discussed. MAJOR CONCLUSION: Stimulation of IDE synthesis is effective for the clinical treatment of metabolic diseases including AD and T2D. GENERAL SIGNIFICANCE: Cyclo-Z might be the effective treatment of AD and T2D by stimulating IDE synthesis.

Micro-CT in the Assessment of Pediatric Renal Osteodystrophy by Bone Histomorphometry.

BACKGROUND AND OBJECTIVES: Computed tomography (CT) measurements can distinguish between cortical and trabecular bone density in vivo. High-resolution CTs assess both bone volume and density in the same compartment, thus potentially yielding information regarding bone mineralization as well. The relationship between bone histomorphometric parameters of skeletal mineralization and bone density from microcomputed tomography (µCT) measurements of bone cores from patients on dialysis has not been assessed. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Bone cores from 68 patients with ESRD (age =13.9±0.5 years old; 50% men) and 14 controls (age =15.3±3.8 years old; 50% men) obtained as part of research protocols between 1983 and 2006 were analyzed by bone histomorphometry and µCT. RESULTS: Bone histomorphometric diagnoses in the patients were normal to high bone turnover in 76%, adynamic bone in 13%, and osteomalacia in 11%. Bone formation rate did not correlate with any µCT determinations. Bone volume measurements were highly correlated between bone histomorphometry and µCT (bone volume/tissue volume between the two techniques: r=0.70; P<0.001, trabecular thickness and trabecular separation: r=0.71; P<0.001, and r=0.56; P<0.001, respectively). Osteoid accumulation as determined by bone histomorphometry correlated inversely with bone mineral density as assessed by µCT (osteoid thickness: r=-0.32; P=0.01 and osteoid volume: r=-0.28; P=0.05). By multivariable analysis, the combination of bone mineral density and bone volume (as assessed by µCT) along with parathyroid hormone and calcium levels accounted for 38% of the variability in osteoid volume (by histomorphometry). CONCLUSIONS: Measures of bone volume can be accurately assessed with µCT. Bone mineral density is lower in patients with excessive osteoid accumulation and higher in patients with adynamic, well mineralized bone. Thus, bone mineralization may be accurately assessed by µCT of bone biopsy cores. Additional studies are warranted to define the value of high-resolution CT in the prediction of bone mineralization in vivo.

Induction of CXC chemokines in human mesenchymal stem cells by stimulation with secreted frizzled-related proteins through non-canonical Wnt signaling.

AIM: To investigate the effect of secreted frizzled-related proteins (sFRPs) on CXC chemokine expression in human mesenchymal stem cells (hMSCs). METHODS: CXC chemokines such as CXCL5 and CXCL8 are induced in hMSCs during differentiation with osteogenic differentiation medium (OGM) and may be involved in angiogenic stimulation during bone repair. hMSCs were treated with conditioned medium (CM) from L-cells expressing non-canonical Wnt5a protein, or with control CM from wild type L-cells, or directly with sFRPs for up to 10 d in culture. mRNA expression levels of both CXCL5 and CXCL8 were quantitated by real-time reverse transcriptase-polymerase chain reaction and secreted protein levels of these proteins determined by ELISA. Dose- (0-500 ng/mL) and time-response curves were generated for treatment with sFRP1. Signal transduction pathways were explored by western blot analysis with pan- or phosphorylation-specific antibodies, through use of specific pathway inhibitors, and through use of siRNAs targeting specific frizzled receptors (Fzd)-2 and 5 or the receptor tyrosine kinase-like orphan receptor-2 (RoR2) prior to treatment with sFRPs. RESULTS: CM from L-cells expressing Wnt5a, a non-canonical Wnt, stimulated an increase in CXCL5 mRNA expression and protein secretion in comparison to control L-cell CM. sFRP1, which should inhibit both canonical and non-canonical Wnt signaling, surprisingly enhanced the expression of CXCL5 at 7 and 10 d. Dickkopf1, an inhibitor of canonical Wnt signaling prevented the sFRP-stimulated induction of CXCL5 and actually inhibited basal levels of CXCL5 expression at 7 but not at 10 d post treatment. In addition, all four sFRPs isoforms induced CXCL8 expression in a dose- and time-dependent manner with maximum expression at 7 d with treatment at 150 ng/mL. The largest increases in CXCL5 expression were seen from stimulation with sFRP1 or sFRP2. Analysis of mitogen-activated protein kinase signaling pathways in the presence of OGM showed sFRP1-induced phosphorylation of extracellular signal-regulated kinase (ERK) (p44/42) maximally at 5 min after sFRP1 addition, earlier than that found in OGM alone. Addition of a phospholipase C (PLC) inhibitor also prevented sFRP-stimulated increases in CXCL8 mRNA. siRNA technology targeting the Fzd-2 and 5 and the non-canonical Fzd co-receptor RoR2 also significantly decreased sFRP1/2-stimulated CXCL8 mRNA levels. CONCLUSION: CXC chemokine expression in hMSCs is controlled in part by sFRPs signaling through non-canonical Wnt involving Fzd2/5 and the ERK and PLC pathways.

Constitutive expression of human telomerase enhances the proliferation potential of human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are highly desirable cells for bone engineering due to the inherent multipotent nature of the cells. Unfortunately, there is a high degree of variability, as primary hMSC cultures quickly undergo replicative senescence with loss of proliferative potential as they are continually propagated in cell culture. We sought to reduce the variability of these cells by insertion and expression of human telomerase reverse transcriptase (TERT) to immortalize the cell line. hMSCs were transduced with a lentivirus containing the human TERT gene. The resulting cell line has been propagated through more than 70 population-doubling level (PDL) to date and continues to grow exhibiting the characteristic fibroblastic hMSC phenotype. Expression of TERT mRNA and protein activity was confirmed in the TERT-transduced cells. Mock-transduced hMSCs had almost undetectable levels of TERT mRNA and protein activity and lost proliferation potential at PDL 14. The enhanced growth capacity of the hMSC TERT cells was due to increased cell proliferation and reduced cellular senescence rather than due to inhibition of apoptosis. The multipotent nature of the TERT cells was confirmed by differentiation toward the osteoblastic and adipogenic lineages in vitro. Osteoblastic differentiation was confirmed by both expression of alkaline phosphate and mineral deposition visualized by Alizarin Red staining. Adipogenic differentiation was confirmed by production of lipid droplets, which were detected by Oil Red-O staining. In summary, we have generated a stable hMSC line that can be continually propagated and retains both osteoblastic and adipogenic differentiation potential.

In vitro mineralization of human mesenchymal stem cells on three-dimensional type I collagen versus PLGA scaffolds: a comparative analysis.

BACKGROUND: Development of a tissue engineered bone graft requires efficient bioactivity screening of biomaterials in clinically relevant three-dimensional systems. The authors analyzed the relative osteogenic potential of two three-dimensional biomaterials--type I collagen and poly(L-lactide-co-glycolide) (PLGA)--to support in vitro mineralization of human mesenchymal stem cells. METHODS: Human mesenchymal stem cells were seeded onto three-dimensional PLGA or type I collagen scaffolds; incubated in osteogenic media; and harvested at 1, 4, and 7 days. Messenger RNA expression was analyzed using quantitative real-time reverse-transcriptase polymerase chain reaction for osteogenic (i.e., alkaline phosphatase, osteocalcin, bone sialoprotein, Runx2/core binding factor α-1) and angiogenic (i.e., vascular endothelial growth factor and interleukin-8) markers. Alkaline phosphatase enzyme activity was measured at 4 and 7 days. Mineralization was detected by alizarin red staining and micro-computed tomographic imaging at 8 and 12 weeks. Mineral composition was analyzed by solid-phase nuclear magnetic resonance spectroscopy. RESULTS: Early osteogenic and angiogenic markers, and alkaline phosphatase enzyme activity, were up-regulated on PLGA versus collagen scaffolds. However, long-term mineralization endpoints favored type I collagen. By 8 weeks, human mesenchymal stem cells on collagen exhibited significantly higher mineral density by micro-computed tomographic and alizarin red staining than PLGA scaffolds. Both biomaterials deposited calcium hydroxyapatite as determined by nuclear magnetic resonance spectroscopy. CONCLUSIONS: The authors' findings suggest that despite early PLGA induction of osteogenic gene expression, long-term mineralization occurs earlier and to a greater extent on type I collagen, highlighting collagen as a potential bone tissue engineering scaffold in the human mesenchymal stem cell niche. When screening the relative osteoinductive profiles of three-dimensional bone tissue engineering scaffolds in vitro, the authors recommend including long-term endpoints of osteogenesis.

CXC receptor knockout mice: characterization of skeletal features and membranous bone healing in the adult mouse.

The potential role of CXC chemokines bearing the glu-leu-arg (ELR) motif in bone repair was studied using a cranial defect (CD) model in mice lacking the CXC receptor (mCXCR(-/-) knockout mice), which is homologous to knockout of the human CXC receptor 2 (CXCR2) gene. During the inflammatory stage of bone repair, ELR CXC chemokines are released by inflammatory cells and serve as chemotactic and angiogenic factors. mCXCR(-/-) mice were smaller in weight and length from base of tail to nose tip, compared to WT littermates. DEXA analysis indicated that bone mineral density (BMD), bone mineral content (BMC), total area (TA), bone area (BA), and total tissue mass (TTM) were decreased in the mCXCR(-/-) mice at 6, 12, and 18 weeks of age. Trabecular bone characteristics in mCXCR(-/-) (% bone, connectivity, number, and thickness) were reduced, and trabecular spacing was increased as evidenced by µCT. There was no difference in bone formation or resorption indices measured by bone histomorphometry. Trabecular BMD was not altered. Cortical bone volume, BMD, and thickness were reduced; whereas, bone marrow volume was increased in mCXCR(-/-). Decreased polar moment of inertia (J) in the tibias/femurs suggested that the mCXCR(-/-) long bones are weaker. This was confirmed by three-point bending testing of the femurs. CDs created in 6-week-old male mCXCR(-/-) and WT littermates were not completely healed at 12 weeks; WT animals, however, had significantly more bone in-growth than mCXCR(-/-). New bone sites were identified using polarized light and assessed for numbers of osteocyte (OCy) lacunae and blood vessels (BlV) around the original CD. In new bone, the number of BlV in WT was >2× that seen in mCXCR(-/-). Bone histomorphometry parameters in the cranial defect did not show any difference in bone formation or resorption markers. In summary, studies showed that mCXCR(-/-) mice have (1) reduced weight and size; (2) decreased BMD and BMC; (3) decreased amounts of trabecular and cortical long bone; (4) decreased femur bone strength; and (5) significantly reduced intramembranous bone formation and number of BlV in new calvarial bone during bone repair.

Acidic pH stimulates the production of the angiogenic CXC chemokine, CXCL8 (interleukin-8), in human adult mesenchymal stem cells via the extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and NF-kappaB pathways.

Blood vessel injury results in limited oxygen tension and diffusion leading to hypoxia, increased anaerobic metabolism, and elevated production of acidic metabolites that cannot be easily removed due to the reduced blood flow. Therefore, an acidic extracellular pH occurs in the local microenvironment of disrupted bone. The potential role of acidic pH and glu-leu-arg (ELR(+)) CXC chemokines in early events in bone repair was studied in human mesenchymal stem cells (hMSCs) treated with medium of decreasing pH (7.4, 7.0, 6.7, and 6.4). The cells showed a reciprocal increase in CXCL8 (interleukin-8, IL-8) mRNA levels as extracellular pH decreased. At pH 6.4, CXCL8 mRNA was induced >60x in comparison to levels at pH 7.4. hMSCs treated with osteogenic medium (OGM) also showed an increase in CXCL8 mRNA with decreasing pH; although, at a lower level than that seen in cells grown in non-OGM. CXCL8 protein was secreted into the medium at all pHs with maximal induction at pH 6.7. Inhibition of the G-protein-coupled receptor alpha, G(alphai), suppressed CXCL8 levels in response to acidic pH; whereas phospholipase C inhibition had no effect on CXCL8. The use of specific mitogen-activated protein kinase (MAPK) signal transduction inhibitors indicated that the pH-dependent increase in CXCL8 mRNA is due to activation of ERK and p38 pathways. The JNK pathway was not involved. NF-kappaB inhibition resulted in a decrease in CXCL8 levels in hMSCs grown in non-OGM. However, OGM-differentiated hMSCs showed an increase in CXCL8 levels when treated with the NF-kappaB inhibitor PDTC, a pyrrolidine derivative of dithiocarbamate.

Regulation of proliferation and migration in retinoic acid treated C3H10T1/2 cells by TGF-beta isoforms.

UNLABELLED: Proliferation of mesenchymal precursors of osteogenic and chondrogenic cells and migration of these precursors to repair sites are important early steps in bone repair. Transforming growth factor-beta (TGF-beta) has been implicated in the promotion of bone repair and may have a role in these processes. Three isoforms of TGF-beta, TGF-beta1, -beta2, and -beta3, are expressed in fracture healing, however, their specific roles in the repair process are unknown. Differential actions of the TGF-beta isoforms on early events of bone repair were explored in the multipotent mesenchymal precursor cell line, C3H10T1/2. Cell migration was determined using a modified Boyden chamber in response to concentrations of each isoform ranging from 10(-12) to 10(-9) g/ml. All three isoforms demonstrated a dose-dependent chemotactic stimulation of untreated C3H10T1/2 cells. Checkerboard assays indicated that all three isoforms also stimulated chemokinesis of the untreated cells. C3H10T1/2 cells treated with all-trans-retinoic acid (ATRA) and expressing relatively higher levels of osteoblastic gene markers such as alkaline phosphatase and collagen type I, lower levels of chondrocytic gene markers collagen type II and aggrecan, and unchanged levels of the adipose marker adipsin did not demonstrate significant chemokinesis or chemotaxis in response to TGF-beta1 or -beta3 at concentrations ranging from 10(-12) to 10(-9) g/ml. In the ATRA-treated cells, TGF-beta2 stimulated a significant increase in chemotaxis only at the highest concentration tested. Cell proliferation was assessed by mitochondrial dehydrogenase activity and cell counts at TGF-beta concentrations from 10(-11) to 10(-8) g/ml. None of the TGF-beta isoforms stimulated cell proliferation in untreated or ATRA-treated C3H10T1/2 cells. Analysis of TGF-beta receptors (TGF-betaR1, -betaR2, and -betaR3) showed a 1.6- to 2.8-fold decrease in mRNA expression of these receptors in ATRA-treated cells. IN CONCLUSION: (1) while all three TGF-beta isoforms stimulate chemotaxis/chemokinesis of multipotent C3H10T1/2 cells, TGF-beta1 and -beta3 do not stimulate chemotaxis in C3H10T1/2 cells treated with ATRA while TGF-beta2 stimulated chemotaxis only at the highest concentration tested. (2) TGF-beta isoforms do not appear to stimulate cell proliferation in C3H10T1/2 cells in either a multipotent state or after ATRA treatment when expressing higher levels of alkaline phosphatase and collagen type I gene markers. (3) Decrease in mRNA expression for TGF-betaR1, -betaR2, and -betaR3 upon ATRA treatment could potentially explain the lack of chemotaxis/chemokinesis in these cells expressing higher levels of alkaline phosphatase and collagen type I.