Smurf2 in senescence, aging and diseases

Smurf2 is a fascinating gene and enzyme that plays a number of key roles throughout life in people, ranging from roles in embryonic development and stem cell differentiation to ones relating to cell senescence and accelerated (or delayed) aging.  It is also implicated in cancers and osteoarthritis.   I strive here to summarize some of the key properties of this substance and point out why it is particularly interesting from the viewpoint of aging. I was made aware of the importance of this substances by a presentation by Hong Zhang at the recent Ellison Medical Foundation’s Colloquium on the Biology of Aging.  Zhang is a researcher at the University of Massachusetts Medical School

What is Smurf2?

The biochemical activities and genetic activation pathway related to Smurf2 are very complex.  In super-simplified terms, Smurf2 has a lot to do with key processes that go in cells including cell reproduction, apoptosis and differentiation.    Smurf2 stands for SMAD specific E3 ubiquitin protein ligase 2, an enzyme that in humans is encoded by the SMURF2 gene.  One of the first documents discussing Smurf2, published in 2000, was Smurf2 is a ubiquitin E3 ligase mediating proteasome-dependent degradation of Smad2 in transforming growth factor-beta signaling. Decoded, this means that Smurf2 links up with ubiquitin (a small regulatory protein found in almost all cells with nuclei that directs proteins for breakdown and recycling) for breakdown of Smad2  in the proteasomes (large protein complexes in cells that break down and recycle unwanted proteins) as part of transforming growth factor-beta  (a protein that controls proliferation, cellular differentiation, and other functions in most cells) signaling.  “SMAD2 mediates the signal of the transforming growth factor (TGF)-beta, and thus regulates multiple cellular processes, such as cell proliferation, apoptosis, and differentiation(ref)” Whew! 

The 2001 document Regulation of Smad degradation and activity by Smurf2, an E3 ubiquitin ligase expands further on the early understanding of the actions of Smurf2.  “Smad proteins are key intracellular signaling effectors for the transforming growth factor-beta superfamily of peptide growth factors. Following receptor-induced activation, Smads move into the nucleus to activate transcription of a select set of target genes. The activity of Smad proteins must be tightly regulated to exert the biological effects of different ligands in a timely manner. Here, we report the identification of Smurf2, a new member of the Hect family of E3 ubiquitin ligases.  Smurf2 selectively interacts with receptor-regulated Smads and preferentially targets Smad1 for ubiquitination and proteasome-mediated degradation.”       

Telomere length attrition, Smurf2 and cell senescence

There are a number of publications further detailing actions of Smurf2, but I am focusing this discussion on longevity-related issues so I move now to a December 2004 publication co-authored by Zhang Smurf2 up-regulation activates telomere-dependent senescence.  “Progressive telomere shortening activates replicative senescence, which prevents somatic cells from being propagated indefinitely in culture. The limitation of proliferative capacity imposed by replicative senescence is thought to contribute to both organismal aging and the prevention of tumor development. Here we report that up-regulation of Smurf2, an E3 ubiquitin ligase previously implicated in TGF-β signaling, is a specific consequence of telomere attrition in human fibroblasts and that such up-regulation is sufficient to produce the senescence phenotype.”  In other words, telomere attrition leads to upregulation of Smurf2 which in turn drives the cell into senescence.  Smurf2 upregulation is an intermediary between telomere attrition and cell senescence.

Telomere attrition can be due to DNA damage, oxidative stress, oncogenic activation or aging.  Continuing to quote from the same 2004 article, “We show that the senescence-inducing actions of Smurf2 occur in the absence of detectable DNA damage or stress response, that Smurf2’s effects require a novel function distinct from its E3 activity, that Smurf2 recruits the Rb and p53 pathways for senescence induction, and that while p21 is elevated by Smurf2, Smurf2-mediated senescence is independent of p21. Smurf2 is the first gene found to be both up-regulated by telomere attrition and sufficient to induce senescence.”   

Expression of Smurf2 in fibroblasts appears to depend on telomere attrition, not on how many times a cell reproduces.  “Importantly, fibroblasts immortalized by adventitious expression of hTERT and analyzed after multiple passages in culture showed no increase in Smurf2 expression (Fig. 1C), indicating that up-regulation of Smurf2 is not the result of extended cell passage per se but, rather, is a consequence of telomere shortening.”  And exposing immortalized fibroblasts with long and stable telomeres to Smurf2 drives them directly into a senescent state.  “Smurf2 expression produces senescence in hTERT-immortalized cells —  Expression of hTERT in primary cultures of human fibroblasts precludes the progressive shortening of telomeres that activates events leading to senescence, resulting in the immortalization of cell populations (Bodnar et al. 1998; Vaziri and Benchimol 1998; Dickson et al. 2000). In strong support of the conclusion that Smurf2 expression is sufficient to produce the senescence phenotype, we found that adventitious expression of Smurf2 to the level normally observed during replicative senescence induced by telomere attrition (cf. Figs. ​Figs.5A5A and ​and1B) reversed hTERT-mediated immortalization of human fibroblasts. — Collectively, our findings support the argument that Smurf2 up-regulation mediates one of the multiple cellular pathways that have been proposed to lead to senescence (Pereira-Smith and Smith 1988).” 

These findings are clearly relevant to the 12^th theory of aging laid out in my treatise Telomere Shortening and Damage.   Long telomeres and even extraordinary expression of telomerase cannot protect a cell from senescence if Smurf2 is also strongly present in that cell. 

Zhang has been concerned with the overall process of cell senescence as well as with the specific role of Smurf2 and has generated a number of publications  on the topic including a comprehensive 2007review paper Molecular signaling and genetic pathways of senescence: Its role in tumorigenesis and aging.  His discussions in that paper of senescence and aging, senescence as a tumor suppression mechanism, and senescence and tissue microenvironment are worth reading though there has been much relevant subsequent research.   

The 2008 paper Suppression of human tumor cell proliferation by Smurf2-induced senescence, again co-authored by Zhang, continues to tell the story, this time extending the research from fibroblasts cells to a wide variety of cell types including cancer cells.    “Here we report that Smurf2 up-regulation induced senescence in a wide variety of human cell types, including highly neoplastic cell lines. Consistent with our previous findings, the ability of Smurf2 to arrest cell proliferation did not require its ubiquitin ligase activity. Furthermore, expression of the cyclin-dependent kinase inhibitor p21 was increased in tumor cells undergoing Smurf2-induced senescence, and such increase occurred independently of the transactivation function of p53. Our results, which reveal a previously unsuspected tumor suppression function for Smurf2-induced senescence, suggest that modulation of Smurf2 action may be a useful strategy for inhibition of cancer cell growth.”   

From an evolutionary viewpoint, it appears that one role of Smurf2 is protection against cancers.  Cells experiencing telomere attrition are driven into senescence by Smurf2 rather than being exposed to the possibility of oncogenesis due to DNA damage that could be incurred in further cycles of replication.  The upside of cell senescence is limiting the accumulation of additional DNA mutations and limiting the population of cells at risk for neoplastic transformation.  The downside is limiting the renewal capacity of stem and progenitor cells and negative changes in gene expression and cell secretions.  Senescent cells make bad neighbors and contribute to organismal aging.  Quoting from my treatise  “It appears that cellular senescence initiates a self-amplifying cycle between mitochondrial and telomeric DNA damage.  The telomere shortening theory of aging suggests that when a substantial number of cells in an organ approach the Hayflick limit and cell senescence, integrity of that organ can no longer be assured and that virtually all of the conditions and diseases of old age are thus traceable to cell senescence.” 

At his presentation at the Ellison colloquium, Zhang described another round of Smurf2 research, this time based on breeding a new strain of Smurf2-knockout mice.  In culture, the cells in these mice generally survived longer with more population doublings,  and were more prone to turning cancerous when compared with similar cells from  normal; “wild type” mice. The Smurf2 knockout mice showed a significant increase in B cell proliferation.  Conclusions of the slide presentation were “1.  Smurf2-deficient MEFs exhibit delayed senescence and enhanced potential to immortalize in culture, 2.  Smurf2 deficient mice develop tumors including lymphomas, soft tissue sarcomas, small intestine andocarcinomas and hepatocellular carcinomas, suggesting that Smurf2 is a tumor suppressor, and 3. Smurf2 deficient mice have increased bone marrow and LT-HSC populations, suggesting a beneficial effect of Smurf2 deficiency during aging.”   

So, we have another example of a familiar story, in aging Smurf2 is strongly protective against cancers but at a cost of limiting stem and progenitor cell differentiation and the increased life span that would result from this.  Smurf2 is similar to P16/Ink4a in this regard.  As I said in my treatise, “Unfortunately there is a paradox in that the same mechanisms that promote neurogenesis, like expression of Bcl-2 and NF-kappaB, can also promote carcinogenesis(ref)., The Ink4a proteins which are increasingly active with age suppress those mechanisms leading to increased protection against cancers with age, but at the cost of decreased neurogenesis and decreased proliferation of other somatic stem cell types. Sorting out the differences between the biomolecular programs that promote stem cell expression and the programs that promote cancers, assuming there are some differences, is a major challenge that must be overcome if substantial life extension is to be made possible.”  Smurf2 is another Dr Jekyll and Mr. Hyde protein. 

Smurf2 and cancers 

A number of past and relatively recent papers point out ugly things Smurf2 does in cancers, like the 2002 publication High-level expression of the Smad ubiquitin ligase Smurf2 correlates with poor prognosis in patients with esophageal squamous cell carcinoma.  The 2009 publication Smad ubiquitination regulatory factor 2 promotes metastasis of breast cancer cells by enhancing migration and invasiveness states “Overexpression of Smurf2 promotes metastasis in a nude mouse model and increases migration and invasion of breast cancer cells. Moreover, expression of Smurf2CG, an E3 ligase-defective mutant of Smurf2, suppresses the above metastatic behaviors. These results establish an important role for Smurf2 in breast cancer progression and indicate that Smurf2 is a novel regulator of breast cancer cell migration and invasion.” 

Not all actions of Smurf2 are negative with respect to cancers, however.  The 2008 publication Smurf2 induces ubiquitin-dependent degradation of Smurf1 to prevent migration of breast cancer cells states “In the present study, we show the post-translational regulation of Smurf1 by Smurf2 and the functional differences between Smurf1 and Smurf2 in the progression of breast cancer cells. Smurf2 interacted with Smurf1 and induced its ubiquitination and degradation, whereas Smurf1 failed to induce degradation of Smurf2. Knockdown of Smurf2 in human breast cancer MDA-MB-231 cells resulted in increases in the levels of Smurf1 protein, and enhancement of cell migration in vitro and bone metastasis in vivo. — These results indicate that two related E3 ubiquitin ligases, Smurf1 and Smurf2, act in the same direction in TGF-beta family signaling but play opposite roles in cell migration.” 

Smurf2 and Osteoarthritis 

Smurf2 also plays a negative role in certain other age-related disease processes, specifically, osteoarthritis.  The 2009 publication Smurf2 induces degradation of GSK-3beta and upregulates beta-catenin in chondrocytes: a potential mechanism for Smurf2-induced degeneration of articular cartilage has to say: “We have previously demonstrated that Smurf2 is highly expressed in human osteoarthritis (OA) tissue, and overexpression of Smurf2 under the control of the type II collagen promoter (Col2a1) induces an OA-like phenotype in aged Col2a1-Smurf2 transgenic mice, suggesting that Smurf2 is located upstream of a signal cascade which initiates OA development. — Furthermore, we discovered that ectopically expressed Smurf2 interacted with GSK-3beta and induced its ubiquitination and subsequent proteasomal degradation, and hence upregulated beta-catenin in Col2a1-Smurf2 transgenic chondrocytes ex vivo. It is therefore likely that Smurf2-mediated upregulation of beta-catenin through induction of proteasomal degradation of GSK-beta in chondrocytes may activate articular chondrocyte maturation and associated alteration of gene expression, the early events of OA.”  The 2010 publication β-catenin, cartilage, and osteoarthritis states flatly “Overexpression of Smurf2, an E3 ubiquitin ligase, also induces an OA-like phenotype through upregulation of β-catenin signaling.” 

From the Arthritis Foundation’s website:  “A new clinical trial seeks to predict who is most likely to experience osteoarthritis, and to test whether an experimental treatment can prevent it altogether. Physicians are setting their sights on people who sustain a knee injury, seeking to understand why nearly half of them will later go on to develop osteoarthritis. — Initial research has shown an enzyme that controls the response of cells to growth factors may in fact be a major cause of osteoarthritis. The enzymes are called “Smad Ubiquitination Regulatory Factors,” or smurfs; but unlike the small, loveable blue cartoon characters, researchers believe that a particular form of these regulatory enzymes, smurf2, might be responsible for America’s leading cause of disability. — We believe that smurf2 controls whether or not a cartilage cell matures and calcifies into hard bone, which is a very good thing when ‘turned on’ in those areas of the body where we are supposed to have hard bone,” said Randy Rosier, MD, PhD, professor of Orthopaedics and director of Research Translation in Orthopaedics at the University of Rochester Medical Center in New York. “But when smurf2 is active in joint cartilage, it may set off a chain reaction that leads to the steady deterioration of the smooth gliding surface tissue, or cartilage, which comprises the joint surface. When this occurs, the cartilage breaks down and severely damages the weight-bearing surface of a joint. Or, put another way, activation of smurf2 in the joint cartilage appears to significantly contribute to the onset of osteoarthritis.”  The clinical trial referenced is entitled Chondrocyte Maturation and Cartilage Loss Following Meniscal Injury and is currently recruiting participants. 

Smurf2 is another odd-shaped piece of the longevity and health jigsaw puzzle with possible future implications both for treatment of diseases of the aged and pro-longevity interventions.