Roberto Civitelli

Tumor growth and metastases are affected by interactions between tumor and microenvironment cells. We have reported the presence of Sp7 -positive cells with an osteogenic signature in primary mouse breast cancer, where they stimulate tumor growth; and genetic ablation of Cdh 2 (encoding N-cadherin) in these cells enhances their pro-tumorigenic action. To study the molecular mechanisms of this biologic system, we used MC3T3 cells, phenotypically similar to tumor associated osteolineage cells. Ablation of Cdh2 in MC3T3 cells enhances PI3K-Akt-β-catenin signaling in response to transforming growth factor-β1 (Tgf-β1), resulting in increased production of Tgf-β1. Interference with PI3K activity is mediated by N-cadherin binding to PI3K components, p85α and p100, resulting in reduced activation of PI3K-Akt-β-catenin signaling. Downstream, Cdh2 ablation enhances Tgf-β1-induced binding of Sp1 and Lef-1 to the Tgfb1 promoter, leading to increased promoter activity and enhanced Tgf-β1 production. This is associated with miR-21 up-regulation and decreased expression of Pten, a PI3K inhibitor. MC3T3 cells promote growth of breast cancer cells (BCC) when co-cultured in vitro or co-injected in mouse mammary fat pad, and Cdh2 -deficiency enhances this pro-tumorigenic effect. Notably, genetic ablation of the Tgf-β1 receptor subunit, Tgfbr1, in BCC abrogates the pro-tumorigenic action of MC3T3 cells and its enhancement by Cdh2 ablation. Finally, Sp7-driven Tgfbr1 ablation in mice also reduces the growth of BCC in the mammary fat pad. Thus, autocrine Tgf-β1 production via PI3K-Akt-β-catenin signaling is a key mechanism by which osteolineage cells promote BCC growth, an action restrained by Ncad via interference with PI3K components. Highlights: In tumor-associated osteolineage cells, N-cadherin reduces the growth of breast tumors; it also inhibits Tgf-β1-activated PI3K/AKT/β-catenin signaling Tgf-β1 produced by tumor microenvironment cells stimulates breast cancer cell growth and further autocrine production of Tgf-β1 The anti-tumorigenic action of N-cadherin is mediated by a braking effect on a Tgf-β1-driven, pro-tumorigenic, feed-forward cycle between microenvironment and tumor cells.

Many studies have compared gene expression in young and old samples to gain insights on aging, the primary risk factor for most chronic diseases. However, these studies only identify associations without distinguishing drivers of aging from compensatory geroprotective responses or incidental downstream effects. Here, we introduce a workflow to characterize causal effects of differentially expressed genes on lifespan. First, we performed a meta‐analysis of 25 gene expression datasets comprising samples of various tissues from healthy, untreated adult mammals (humans, dogs, and rodents) at two distinct ages. Genes were ranked by the number of datasets in which they exhibited consistent differential expression with age. The top age‐upregulated genes were TMEM176A, EFEMP1, CP, and HLA‐A; the top age‐downregulated genes were CA4, SIAH, SPARC, and UQCR10. Second, the effects of the top ranked genes on lifespan were measured by applying post‐developmental RNA interference of the corresponding ortholog in Caenorhabditis elegans . Out of 10 age‐upregulated and 9 age‐downregulated genes that were tested, two age‐upregulated genes ( csp‐3 /CASP1 and spch‐2 /RSRC1) and four age‐downregulated genes ( C42C1.8 /DIRC2, ost‐1 /SPARC, fzy‐1 /CDC20, and cah‐3 /CA4) produced significant and reproducible lifespan extension. Notably, the data do not suggest that the direction of differential expression with age is predictive of the effect on lifespan. Our study provides novel insight into the relationship between differential gene expression and aging phenotypes, pilots an unbiased workflow that can be easily repeated and expanded, and pinpoints six genes with evolutionarily conserved, causal roles in the aging process for further study.

Type 2 diabetes (T2D) and obesity (OB) are associated with chronic low-grade inflammation and increased fracture risk. In vitro studies showed that inflammation induces bone erosion and inhibits bone formation by increasing Wnt canonical pathway inhibitors. However, the impact of inflammation on Wnt pathway regulation and bone quality in T2D and OB remains unclear. To this end, we studied 63 postmenopausal women (age >65 years) undergoing hip replacement for osteoarthritis. Among these women, 19 had T2D and OB (HbA1c 6.8±0.79%; BMI 29.9±5.2 kg/m2), 17 had OB but they were normoglycemic (BMI 32.5±5.4 kg/m2), and 27 served as controls (BMI 23.1±5.5 kg/m2). Serum inflammatory cytokines by automated immunoassay (ELLA), revealed higher TNF-α (p=0.0084) and lower adiponectin (p=0.0402) in T2D, and higher IL-6 (p=0.0003) levels in OB vs controls. Gene expression analysis of trabecular bone showed increased TNF-α (p=0.0019) and SFRP5 (p=0.0084) in T2D vs controls. IL-10 was lower in both T2D (p=0.0285), and OB (p=0.0324), while adiponectin (ADIPOQ) was only lower in T2D (p=0.0041) vs controls. Interestingly, the Wnt inhibitor SOST was higher in T2D (p<0.0001) and OB (p<0.0001) vs controls. Conversely, WNT10B mRNA levels were lower in T2D (p=0.0071) and in OB (p=0.0196) vs controls, while LEF-1 were only lower in T2D (p=0.0009). WNT5A (p=0.0025) and GSK3β (p=0.0003) mRNA levels were higher only T2D vs controls. Importantly, TNF-α mRNA levels positively correlated with SOST (r=0.5121, p=0.0002), WNT5A (r=0.3227, p=0.0396) and GSK3β (r=0.3789, p=0.0146) mRNA levels, but negatively correlated with WNT10B (r=0.3844, p=0.0188) and LEF-1(r=-0.3310, p=0.0322) mRNA levels. Conversely, IL-10 was negatively correlated with SOST mRNA levels (r=0.3100, p=0.0457). ADIPOQ was negatively correlated with SOST (r=-0.3864, p=0.0105) and WNT5A (r=-0.3025, p=0.0515) mRNA levels. Moreover, SFRP5 was negatively correlated with LEF-1 mRNA levels (r=0.3991, p=0.0131). Finally, serum levels of TNF-α (r=-0.3473, p=0.0352) and IL-6 (r=-0.3777, p=0.0302) negatively correlated with Young’s Modulus, an index of bone strength. These findings suggest that increased inflammation in bone of subjects with T2D and obesity is negatively associated with Wnt pathway and bone strength, shedding light on pathophysiology of bone impairment in T2D and obesity.

Type 2 diabetes (T2D) and obesity (OB) are associated with chronic low-grade inflammation and increased fracture risk. In vitro studies showed that inflammation induces bone erosion and inhibits bone formation by increasing Wnt canonical pathway inhibitors. However, the impact of inflammation on Wnt pathway regulation and bone quality in T2D and OB remains unclear. To this end, we studied 63 postmenopausal women (age >65 years) undergoing hip replacement for osteoarthritis. Among these women, 19 had T2D and OB (HbA1c 6.8±0.79%; BMI 29.9±5.2 kg/m2), 17 had OB but they were normoglycemic (BMI 32.5±5.4 kg/m2), and 27 served as controls (BMI 23.1±5.5 kg/m2). Serum inflammatory cytokines by automated immunoassay (ELLA), revealed higher TNF-α (p=0.0084) and lower adiponectin (p=0.0402) in T2D, and higher IL-6 (p=0.0003) levels in OB vs controls. Gene expression analysis of trabecular bone showed increased TNF-α (p=0.0019) and SFRP5 (p=0.0084) in T2D vs controls. IL-10 was lower in both T2D (p=0.0285), and OB (p=0.0324), while adiponectin (ADIPOQ) was only lower in T2D (p=0.0041) vs controls. Interestingly, the Wnt inhibitor SOST was higher in T2D (p<0.0001) and OB (p<0.0001) vs controls. Conversely, WNT10B mRNA levels were lower in T2D (p=0.0071) and in OB (p=0.0196) vs controls, while LEF-1 were only lower in T2D (p=0.0009). WNT5A (p=0.0025) and GSK3β (p=0.0003) mRNA levels were higher only T2D vs controls. Importantly, TNF-α mRNA levels positively correlated with SOST (r=0.5121, p=0.0002), WNT5A (r=0.3227, p=0.0396) and GSK3β (r=0.3789, p=0.0146) mRNA levels, but negatively correlated with WNT10B (r=0.3844, p=0.0188) and LEF-1(r=-0.3310, p=0.0322) mRNA levels. Conversely, IL-10 was negatively correlated with SOST mRNA levels (r=0.3100, p=0.0457). ADIPOQ was negatively correlated with SOST (r=-0.3864, p=0.0105) and WNT5A (r=-0.3025, p=0.0515) mRNA levels. Moreover, SFRP5 was negatively correlated with LEF-1 mRNA levels (r=0.3991, p=0.0131). Finally, serum levels of TNF-α (r=-0.3473, p=0.0352) and IL-6 (r=-0.3777, p=0.0302) negatively correlated with Young’s Modulus, an index of bone strength. These findings suggest that increased inflammation in bone of subjects with T2D and obesity is negatively associated with Wnt pathway and bone strength, shedding light on pathophysiology of bone impairment in T2D and obesity.

Autosomal dominant Fanconi syndrome due to a GATM variant (GATM‐FS), causes accumulation of misfolded arginine‐glycine amidinotransferase (AGAT) in proximal renal tubules leading to cellular injury. GATM‐FS presents during childhood and progresses to end‐stage kidney disease (ESKD) in adults. We study creatine metabolism in two individuals of unrelated families with a known GATM variant and the effect of creatine supplementation in kidney organoids. Plasma and urine metabolites were measured by mass spectrometry. Brain creatine was assessed by magnetic resonance spectroscopy (MRS). Guanidinoacetate (GAA) synthesis by the AGAT mutant was measured in patient‐derived immortalized lymphocytes using stable isotopes of arginine and glycine. The effect of creatine on GATM expression was assessed in human kidney cells and organoids. Several family members from two unrelated families were diagnosed with Fanconi syndrome and had the c.1022C>T (p. P341L) variant in GATM. Two affected individuals in both families had moderately reduced plasma GAA levels. In comparison to wild‐type cells, GAA synthesis by patient‐derived GATM^P341L+/− lymphoblastoid cell lines (LCL) was reduced, but not absent as in GATM cells from a patient with creatine deficiency syndrome. In vitro studies on human kidney organoids revealed reduced AGAT expression after treatment with creatine. Finally, we showed in one patient that creatine supplementation (5 g daily) substantially increased plasma creatine levels. We report low plasma and urine GAA in patients with autosomal dominant GATM‐FS and show that creatine downregulates AGAT in human kidney cells.

Many studies have compared gene expression in young and old samples to gain insights on aging, the primary risk factor for most major chronic diseases. However, these studies only describe associations, failing to distinguish drivers of aging from compensatory geroprotective responses and incidental downstream effects. Here, we introduce a workflow to characterize the causal effects of differentially expressed genes on lifespan. First, we performed a meta-analysis of 25 gene expression datasets comprising samples of various tissues from healthy, untreated adult mammals (humans, dogs, and rodents) at two distinct ages. We ranked each gene according to the number of distinct datasets in which the gene was differentially expressed with age in a consistent direction. The top age-upregulated genes were TMEM176A, EFEMP1, CP, and HLA-A; the top age-downregulated genes were CA4, SIAH, SPARC, and UQCR10. Second, the effects of the top ranked genes on lifespan were measured by applying post-developmental RNA interference of the corresponding ortholog in the nematode C. elegans (two trials, with roughly 100 animals per genotype per trial). Out of 10 age-upregulated and 9 age-downregulated genes that were tested, two age-upregulated genes ( csp-3 /CASP1 and spch-2 /RSRC1) and four age-downregulated genes ( C42C1.8 /DIRC2, ost-1 /SPARC, fzy-1 /CDC20, and cah-3 /CA4) produced significant and reproducible lifespan extension. Notably, the data do not suggest that the direction of differential expression with age is predictive of the effect on lifespan. Our study provides novel insight into the relationship between differential gene expression and aging phenotypes, pilots an unbiased workflow that can be easily repeated and expanded, and pinpoints six genes with evolutionarily conserved, causal roles in the aging process for further study. Graphical

Type 2 diabetes (T2D) is associated with higher fracture risk, despite normal or high bone mineral density. We reported that bone formation genes (SOST and RUNX2) and advanced glycation end-products (AGEs) were impaired in T2D. We investigated Wnt signaling regulation and its association with AGEs accumulation and bone strength in T2D from bone tissue of 15 T2D and 21 non-diabetic postmenopausal women undergoing hip arthroplasty. Bone histomorphometry revealed a trend of low mineralized volume in T2D (T2D 0.249% [0.156–0.366]) vs non-diabetic subjects 0.352% [0.269–0.454]; p=0.053, as well as reduced bone strength (T2D 21.60 MPa [13.46–30.10] vs non-diabetic subjects 76.24 MPa [26.81–132.9]; p=0.002). We also showed that gene expression of Wnt agonists LEF-1 (p=0.0136) and WNT10B (p=0.0302) were lower in T2D. Conversely, gene expression of WNT5A (p=0.0232), SOST (pGSK3B (p=0.0456) were higher, while collagen (COL1A1) was lower in T2D (p=0.0482). AGEs content was associated with SOST and WNT5A (r=0.9231, pLEF-1 and COL1A1 (r=–0.7500, p=0.0255; r=–0.9762, p=0.0004). SOST was associated with glycemic control and disease duration (r=0.4846, p=0.0043; r=0.7107, p=0.00174), whereas WNT5A and GSK3B were only correlated with glycemic control (r=0.5589, p=0.0037; r=0.4901, p=0.0051). Finally, Young’s modulus was negatively correlated with SOST (r=−0.5675, p=0.0011), AXIN2 (r=−0.5523, p=0.0042), and SFRP5 (r=−0.4442, p=0.0437), while positively correlated with LEF-1 (r=0.4116, p=0.0295) and WNT10B (r=0.6697, p=0.0001). These findings suggest that Wnt signaling and AGEs could be the main determinants of bone fragility in T2D.

Type 2 diabetes (T2D) is associated with higher fracture risk, despite normal or high bone mineral density. We reported that bone formation genes (SOST and RUNX2) and Advanced Glycation End-products (AGEs) were impaired in T2D. We investigated Wnt signaling regulation and its association with AGEs accumulation and bone strength in T2D from bone tissue of 15 T2D and 21 non-diabetic postmenopausal women undergoing hip arthroplasty. Bone histomorphometry revealed a trend of low mineralized volume in T2D [(T2D 0.249% (0.156-0.366) vs non-diabetic subjects 0.352% (0.269-0.454); p=0.053)], as well as reduced bone strength [T2D 21.60 MPa (13.46-30.10) vs non-diabetic subjects 76.24 MPa (26.81-132.9); p=0.002]. We also showed that gene expression of Wnt agonists LEF-1 (p=0.0136) and WNT10B (p=0.0302) were lower in T2D. Conversely, gene expression of WNT5A (p=0.0232), SOST (p<0.0001) and GSK3B (p=0.0456) were higher, while collagen (COL1A1) was lower in T2D (p=0.0482). AGEs content was associated with SOST and WNT5A (r=0.9231, p<0.0001; r=0.6751, p=0.0322), but inversely correlated with LEF-1 and COL1A1 (r= -0,7500, p=0.0255; r= -0,9762, p=0.0004). SOST was associated with glycemic control and disease duration (r=0.4846, p=0.0043; r=0.7107, p=0.00174), whereas WNT5A and GSK3B were only correlated with glycemic control (r=0.5589, p=0.0037; r=0.4901, p=0.0051). Finally, Young’s Modulus was negatively correlated with SOST (r=-0.5675, p=0.0011), AXIN2 (r=-0.5523, p=0.0042) and SFRP5 (r=-0.4442, p=0.0437), while positively correlated with LEF -1 (r=0.4116, p=0.0295) and WNT10B (r=0.6697, p=0.0001). These findings suggest that Wnt signaling, and AGEs could be the main determinants of bone fragility in T2D.

Connexin43 (Cx43) is the most abundant gap junction protein present in the mesenchymal lineage. In mature adipocytes, Cx43 mediates white adipose tissue (WAT) beiging in response to cold exposure and maintains the mitochondrial integrity of brown adipose tissue (BAT). We found that genetic deletion of Gja1 (Cx43 gene) in cells that give rise to chondro-osteogenic and adipogenic precursors driven by the Dermo1 / Twist2 promoter led to lower body adiposity and partial protection against the weight gain and metabolic syndrome induced by a high-fat diet (HFD) in both sexes. These protective effects were related to increased locomotion, fuel utilization, energy expenditure, nonshivering thermogenesis, and better glucose tolerance in conditionally Gja1 -ablated mice. Accordingly, Gja1 -mutant mice exhibited reduced adipocyte hypertrophy, partially preserved insulin sensitivity, increased BAT lipolysis, and decreased whitening under HFD. This metabolic phenotype was not reproduced with more restricted Gja1 ablation in differentiated adipocytes, suggesting that Cx43 in adipocyte progenitors or other targeted cells restrains energy expenditures and promotes fat accumulation. These results reveal what we believe is a hitherto unknown action of Cx43 in adiposity, and offer a promising new pharmacologic target for improving metabolic balance in diabetes and obesity.